Markers for prenatal diagnosis and monitoring

ABSTRACT

Methods and kits are provided for diagnosing, monitoring, or predicting preeclaimpsia in a pregnant woman, trisomy 18 and trisomy 21 in a fetus, as well as for detecting pregnancy in a woman, by quantitatively measuring in the maternal blood the amount of one or more RNA species derived from a set of genetic loci and comparing the amount of the RNA species with a standard control.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. patent application Ser. No.60/663,293, filed Mar. 18, 2005, the contents of which are hereinincorporated by reference in the entirety.

BACKGROUND OF THE INVENTION

Prenatal diagnosis has been routinely conducted using cells isolatedfrom the fetus through procedures such as chorionic villus sampling(CVS) or amniocentesis. These conventional methods are, however,invasive and present an appreciable risk to both the mother and thefetus despite most careful handling (Tabor et al., Lancet 1:1287-1293,1986).

Alternatives to these invasive approaches have been developed forprenatal screening, e.g., to detecting fetal abnormalities, followingthe discoveries that several types of fetal cells can be found inmaternal circulation (Johansen et al., Prenat. Diagn. 15:921-931, 1995)and more importantly, circulating cell-free fetal DNA can be detected inmaternal plasma and serum (Lo et al., Lancet 350:485-487, 1997). Theamount of fetal DNA in maternal blood has been shown to be sufficientfor genetic analysis without complex treatment of the plasma or serum,in contrast to the necessary steps for isolating and enriching fetalcells. Fetal rhesus D (RhD) genotyping (Lo et al., N. Engl. J. Med.339:1734-1738, 1998), fetal sex determination (Lo et al., Hum. Genet.90:483-488, 1993), and diagnosis of several fetal disorders (Amicucci etal., Clin. Chem. 46:301-302, 2000; Saito et al., Lancet 356:1170, 2000;and Chiu et al., Lancet 360:998-1000, 2002) have since been achieved bydetecting fetal DNA in maternal blood using a polymerase chain reaction(PCR)-based technique.

In addition, quantitative abnormalities of fetal DNA in maternalplasma/serum have also been reported in preeclampsia (Lo et al., Clin.Chem. 45:184-188, 1999 and Zhong et al., Am. J. Obstet. Gynecol.184:414-419, 2001), fetal trisomy 21 (Lo et al., Clin. Chem.45:1747-1751, 1999 and Zhong et al. Prenat. Diagn. 20:795-798, 2000) andhyperemesis gravidarum (Sekizawa et al., Clin. Chem. 47:2164-2165,2001). Detection of fetal nucleic acid in maternal blood for prenatalgenetic analysis is also disclosed in U.S. Pat. No. 6,258,540.

When analyzing fetal DNA, investigators have often used Y chromosomalmarkers, which are only present in male fetuses, as a fetal-specificmarker. This approach has limited the application of this technology tothe 50% of pregnant women who are carrying male fetuses. Further, theuse of other genetic polymorphisms has also increased the complexity offetal DNA-based analyses. The discovery of fetal RNA in maternal plasmaoffers a possible new approach that circumvents these limitations (Poonet al., Clin. Chem. 46:1832-1834, 2000).

More recently, U.S. patent application Ser. No. 09/876,005 disclosesnon-invasive techniques based on detection of fetal/placental RNA inmaternal blood. Further, U.S. patent application Ser. No. 10/759,783discloses certain placental expressed mRNA markers (e.g., humanchorionic gonadotropin β subunit and human corticotropin releasinghormone) that can be used for the detection of pregnancy andpregnancy-related disorders such as preeclampsia, fetal chromosomalaneuploidy, and pre-term labor. Various other RNA species of placentalorigin have also been detected in maternal blood, see, e.g., Oudejans etal., Clin Chem. 2003, 49(9):1445-1449, and Go et al., Clin. Chem. 2004,50(8):1413-1414. The present invention discloses additionalfetal/placenta-derived RNA species, shown in Tables 1-6, that are foundin maternal blood and can be used as markers for detecting pregnancy, orfor genotyping the fetus, or for diagnosing, monitoring, and predictingpreeclampsia and fetal chromosomal aneuploidy such as trisomy 18 andtrisomy 21. Thus, the present invention provides additional tools fornon-invasive prenatal diagnosis and alternative means for pregnancydetection.

BRIEF SUMMARY OF THE INVENTION

In the first aspect, the present invention relates to a method fordiagnosing, monitoring, or predicting preeclampsia in a pregnant woman.This method comprises the following steps: first, quantitativelydetermining the amount of one or more RNA species in a biological sampleobtained from the pregnant woman. The RNA species are independentlyselected from RNA derived from genetic loci consisting of IGFBP3, ABP1,FN1, SLC21A2, KIAA0992, TIMP3, LPL, INHBA, LEP, ADAM12, PAPPA, PAPPA2,and SIGLEC6, and the biological sample is blood, washing from thereproductive tract, urine, saliva, amniotic fluid, or chorionic villus.Second, comparing the amount of the RNA species from the first step to astandard control representing the amount of the RNA species in thecorresponding sample from an average non-preeclamptic pregnant woman. Anincrease or a decrease in the amount of the RNA species from thestandard control indicates preeclampsia or an increased risk ofdeveloping preeclampsia.

In some embodiments, the RNA species is derived from ADAM12, PAPPA2,FN1, INHBA, LEP, or SIGLEC6, and an increase in the amount of the RNAspecies from the standard control indicates preeclampsia or an increasedrisk of developing preeclampsia. In other embodiments, the RNA speciesis derived from PAPPA and a decrease in the amount of the RNA speciesfrom the standard control indicates preeclampsia or an increased risk ofdeveloping preeclaimpsia.

In some embodiments, the first step comprises using a reversetranscriptase polymerase chain reaction (RT-PCR). Optionally, this firststep further comprises using mass spectrometry following RT-PCR. Inother embodiments, the first step comprises using a polynucleotidehybridization method, or using a primer extension reaction.

In some embodiments, the woman being examined is during the firsttrimester of gestation. In other embodiments, the woman is during thesecond or third trimester of gestation.

In some embodiments, the blood is fractionated and the plasma fractionis analyzed. In other embodiments, the blood is fractionated and theserum fraction is analyzed. In some embodiments, the increase in theamount of RNA from the standard control is more than 2-fold. In otherembodiments, the decrease in the amount of RNA from the standard controlis more than 50%.

A kit for diagnosing, monitoring, or predicting preeclampsia in apregnant woman is also provided. This kit comprises the following: (i)PCR primers for quantitatively determining the amount of one or more RNAspecies in a biological sample obtained from the pregnant woman, whereinthe RNA species is independently selected from RNA derived from geneticloci consisting of IGFBP3, ABP1, FN1, SLC21A2, KIAA0992, TIMP3, LPL,INHBA, LEP, ADAM12, PAPPA, PAPPA2, and SIGLEC6, and wherein thebiological sample is blood, washing from the reproductive tract,amniotic fluid, urine, saliva, or chorionic villus; and (ii) a standardcontrol representing the amount of the RNA species in the correspondingsample from an average non-preeclamptic pregnant woman.

In the second aspect, the present invention relates to a method fordetecting the presence of a fetus with trisomy 18 in a pregnant woman.This method comprises the following steps: first, quantitativelydetermining the amount of the RNA species derived from genetic locusRPL17 in a biological sample obtained from the pregnant woman. Thebiological sample is blood, washing from the reproductive tract,amniotic fluid, urine, saliva, or chorionic villus. Second, comparingthe amount of the RPL17 RNA from the first step to a standard controlrepresenting the amount of the RPL17 RNA in the corresponding samplefrom an average pregnant woman carrying a chromosomally normal fetus. Adeviation in the amount of the RNA species from the standard controlindicates an increased risk of having a fetus with trisomy 18.

In some embodiments, an increase in the amount of the RPL17 RNA from thestandard control indicates an increased risk of having a fetus withtrisomy 18; whereas in other cases, a decrease in the amount of theRPL17 RNA from the standard control may indicate an increased risk ofhaving a fetus with trisomy 18.

In some embodiments, the first step comprises using a reversetranscriptase polymerase chain reaction (RT-PCR). Optionally, this firststep further comprises using mass spectrometry following RT-PCR. Inother embodiments, the first step comprises using a polynucleotidehybridization method, or using a primer extension reaction.

In some embodiments, the woman being examined is during the firsttrimester of gestation. In other embodiments, the woman is during thesecond or third trimester of gestation.

In some embodiments, the blood is fractionated and the plasma fractionis analyzed. In other embodiments, the blood is fractionated and theserum fraction is analyzed. In some embodiments, the increase in theamount of RNA from the standard control is more than 2-fold. In otherembodiments, the decrease in the amount of RNA from the standard controlis more than 50%.

A kit for detecting the presence of a fetus with trisomy 18 in apregnant woman is also provided. This kit comprises the following: (i)PCR primers for quantitatively determining the amount of RNA derivedfrom genetic locus RPL17, wherein the biological sample is blood,washing from the reproductive tract, amniotic fluid, urine, saliva, orchorionic villus; and (ii) a standard control representing the amount ofthe RPL17 RNA in the corresponding sample from an average pregnant womancarrying a chromosomally normal fetus.

In a third aspect, the present invention relates to a method fordetecting the presence of a fetus with trisomy 21 in a pregnant woman.The method comprises the following steps of: first, quantitativelydetermining the amount of one or more RNA species in a biological sampleobtained from the pregnant woman. The RNA species is independentlyselected from RNA species derived from genetic loci consisting ofCOL6A1, COL6A2, SOD1, APP, BTG3, ATP5J, ADAMTS1, BACE2, DSCR5, ITSN1,PLAC4, ATP5O, LOC90625, EFEMP1, and TFRC, whereas the biological sampleis blood, washing from the reproductive tract, urine, saliva, amnioticfluid, or chorionic villus. Second, comparing the amount of the RNAspecies from the first step to a standard control representing theamount of the RNA species in the corresponding sample from an averagepregnant woman with a chromosomally normal fetus. An increase or adecrease in the amount of RNA species from the standard controlindicates an increased risk of having a fetus with trisomy 21.

In some embodiments, the RNA species is derived from ADAMTS1, APP,ATP5O, EFEMP1, or TFRC, and an increase in the amount of RNA speciesfrom the standard control indicates an increased risk of having a fetuswith trisomy 21.

In some embodiments, the first step comprises using a reversetranscriptase polymerase chain reaction (RT-PCR). Optionally, this firststep further comprises using mass spectrometry following RT-PCR. Inother embodiments, the first step comprises using a polynucleotidehybridization method, or using a primer extension reaction.

In some embodiments, the woman being examined is during the firsttrimester of gestation. In other embodiments, the woman is during thesecond or third trimester of gestation.

In some embodiments, the blood is fractionated and the plasma fractionis analyzed. In other embodiments, the blood is fractionated and theserum fraction is analyzed. In some embodiments, the increase in theamount of RNA from the standard control is more than 2-fold. In otherembodiments, the decrease in the amount of RNA from the standard controlis more than 50%.

A kit for detecting the presence of a fetus with trisomy 21 in apregnant woman is also provided. This kit comprises the following: (i)PCR primers for quantitatively determining the amount of one or more RNAspecies in a biological sample obtained from the pregnant woman, whereinthe RNA species is independently selected from RNA derived from geneticloci consisting of COL6A1, COL6A2, SOD1, APP, BTG3, ATP5J, ADAMTS1,BACE2, DSCR5, ITSN1, PLAC4, ATP5O, LOC90625, EFEMP1, and TFRC, andwherein the biological sample is blood, washing from the reproductivetract, amniotic fluid, urine, saliva, or chorionic villus; and (ii) astandard control representing the amount of the RNA species in thecorresponding sample from an average pregnant woman carrying achromosomally normal fetus.

In a fourth aspect, the present invention relates to a method fordetecting pregnancy in a woman. The method comprises the following stepsof: first, quantitatively determining the amount of one or more RNAspecies in a biological sample obtained from the woman. The RNA speciesis independently selected from RNA species derived from genetic lociconsisting of COL6A1, COL6A2, SOD1, ATP5O, ADAMTS1, DSCR5, and PLAC4,whereas the biological sample is blood, washing from the reproductivetract, amniotic fluid, urine, saliva, or chorionic villus. Second,comparing the amount of the RNA species from the first step to astandard control representing the amount of the RNA species in thecorresponding sample from an average non-pregnant woman. An increase ora decrease in the amount of RNA species from the standard controlindicates pregnancy.

In some embodiments, the RNA species is derived from COL6A1, COL6A2,ATP5O, or PLAC4, and an increase in the amount of RNA species from thestandard control indicates pregnancy.

In some embodiments, the first step comprises using a reversetranscriptase polymerase chain reaction (RT-PCR). Optionally, this firststep further comprises using mass spectrometry following RT-PCR. Inother embodiments, the first step comprises using a polynucleotidehybridization method, or using a primer extension reaction.

In some embodiments, the woman being examined is during the firsttrimester of gestation. In other embodiments, the woman is during thesecond or third trimester of gestation.

In some embodiments, the blood is fractionated and the plasma fractionis analyzed. In other embodiments, the blood is fractionated and theserum fraction is analyzed. In some embodiments, the increase in theamount of RNA from the standard control is more than 2-fold. In otherembodiments, the decrease in the amount of RNA from the standard controlis more than 50%.

A kit for detecting pregnancy in a woman is also provided. This kitcomprises the following: (i) PCR primers for quantitatively determiningthe amount of one or more RNA species in a biological sample obtainedfrom the pregnant woman, wherein the RNA species is independentlyselected from RNA derived from genetic loci consisting of COL6A1,COL6A2, SOD1, ATP5O, ADAMTS1, DSCR5, and PLAC4, and wherein thebiological sample is blood, washing from the reproductive tract,amniotic fluid, urine, saliva, or chorionic villus; and (ii) a standardcontrol representing the amount of the RNA species in the correspondingsample from an average non-pregnant woman.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Comparison of placental tissue levels of RNA transcripts infirst-trimester trisomy 21 and control pregnancies. (A) ADAMTS1 mRNA.(B) APP mRNA. Each● represents one subject.

FIG. 2. Relative concentrations of placental expressed transcripts inmaternal buffy coat. The lines inside the boxes denote the medians. Theboxes mark the interval between the 25th and 75th percentiles. Thewhiskers denote the interval between the 10th and 90th percentiles. Thefilled circles mark the data points outside the 10th and 90thpercentiles.

FIG. 3. Clearance of placental mRNA from maternal plasma after delivery.Maternal plasma concentrations of (A) COL6A1 mRNA and (B) COL6A2 mRNAbefore delivery and 24 hours after delivery. Each line represents thepaired plasma samples obtained from one subject.

FIG. 4. Comparison of placental tissue levels of RNA transcripts infirst-trimester trisomy 21 and control (normal) pregnancies. (A) EFEMP1mRNA. (B) TFRC mRNA. (C) ATP50 mRNA. Each● represents one subject. (D)Clearance of ATP5O mRNA from maternal plasma 24 hours after delivery.Each line represents the paired plasma samples obtained from onesubject.

FIG. 5. Comparison of placental tissue levels of RNA transcripts inthird-trimester preeclamptic (PET) and control pregnancies. (A) Leptin(LEP) mRNA. (B) SIGLEC6 mRNA. The lines inside the boxes denote themedians. The boxes mark the interval between the 25th and 75thpercentiles. The whiskers denote the interval between the 10th and 90thpercentiles. The filled circles mark the data points outside the 10thand 90th percentiles.

FIG. 6. Comparison of maternal plasma concentrations of (A) Leptin (LEP)mRNA and (B) INHBA mRNA in maternal plasma of preeclamptic and controlpregnancies. The lines inside the boxes denote the medians. The boxesmark the interval between the 25th and 75th percentiles. The whiskersdenote the interval between the 10th and 90th percentiles. The filledcircles mark the data points outside the 10th and 90th percentiles.

FIG. 7. Box plots of PLAC4 mRNA concentrations in the plasma ofnon-pregnant women and women in the first and third trimesters ofpregnancies. The lines inside the boxes denote the medians. The boxesmark the interval between the 25^(th) and 75^(th) percentiles. Thewhiskers denote the interval between the 10^(th) and 90^(th)percentiles. The filled circles mark the data points outside the 10^(th)and 90^(th) percentiles.

FIG. 8. Clearance of PLAC4 mRNA from maternal plasma after delivery.Each line represents the pair of plasma samples obtained from onesubject before and at 24 hours after delivery.

FIG. 9. Comparison of placental tissue levels of RNA transcripts inthird-trimester preeclamptic (PET) and control (normal) pregnancies. (A)LEP mRNA. (B) ADAM12 mRNA. (C) PAPPA mRNA. (D) PAPPA2 mRNA. (E) INHBAmRNA. (F) FN1 mRNA. The lines inside the boxes denote the medians. Theboxes mark the interval between the 25th and 75th percentiles. Thewhiskers denote the interval between the 10th and 90th percentiles. Thefilled circles mark the data points outside the 10th and 90thpercentiles.

DEFINITIONS

The term “an RNA species derived from a genetic locus” as used hereinrefers to a polymer of ribonucleotides that has a sequence correspondingto at least a portion of a pre-selected location in the human genome. An“RNA species” in this application may or may not encode for a proteinproduct, as its sequence may encompass non-coding sequence or includeonly a partial open reading frame.

The term “fetal,” “placental derived,” or “placental expressed” as usedherein describes the origin of certain RNA species that are detectablein a biological sample from a pregnant woman, e.g., blood. In otherwords, a fetal RNA species is one that has been transcribed from a fetalDNA sequence. Furthermore, a placental derived or placental expressedRNA species is one that is found in the placenta and transcribed from afetal DNA sequence.

The term “washing of reproductive tract” as used herein refers to anyliquid or solution that has been collected following the rinse or washof the reproductive tract of a pregnant woman or a woman who is beingtested for possible pregnancy.

The term “preeclampsia” as used herein refers to a condition that occursduring pregnancy, the main symptom of which is various forms of highblood pressure often accompanied by the presence of proteins in theurine and edema (swelling). Preeclampsia, sometimes called toxemia ofpregnancy, is related to a more serious disorder called “eclampsia,”which is preeclampsia together with seizure. These conditions usuallydevelop during the second half of pregnancy (after 20 weeks), thoughthey may develop shortly after birth or before 20 weeks of pregnancy.

The term “primer extension reaction” as used herein refers to anypolymerization process mediated by the action of a nucleotidepolymerase, e.g., a DNA polymerase, by extending a predeterminedpolynucleotide sequence that is at least partially complementary to atemplate sequence under appropriate conditions.

The term “chromosomal aneuploidy” as used herein refers to a state ofchromosomal abnormality where the number of chromosomes is not an exactmultiple of the usual haploid number: frequently, there is either anadditional chromosome or one missing. The most common case of achromosomal aneuploidy is a trisomy, where a single additionalchromosome is present. For example, trisomy 18 is a chromosomalabnormality where a third chromosome 18 is found in a cell, whereas athird chromosome 21 is present in the cells of a patient suffering fromtrisomy 21.

In contrast to aneuploidy, “chromosomally normal” describes the statewhere the number of chromosomes is an exact multiple of the haploidnumber, such as twice the number of chromosomes found in a haploid, andeach chromosome is present in the same number (except the sexchromosomes in the case of, e.g., male humans, where two different sexchromosomes, X and Y, are present at one copy each).

The term “blood” as used herein refers to a blood sample or preparationfrom a pregnant woman or a woman being tested for possible pregnancy.The term encompasses whole blood or any fractions of blood havingvarying concentrations or even no hematopoietic or any other types ofcells or cellular remnants of maternal or fetal origin, includingplatelets. Examples of “blood” include plasma and serum. A blood samplethat is essentially free of cells is also referred to as “acellular,”where generally no platelets are present.

The term “average,” as used in the context of describing a pregnantwoman who is non-preeclamptic, or carries a chromosomally normal fetus,refers to certain characteristics, such as the level of fetal/placentalderived RNA found in maternal blood, that is representative of arandomly selected group of women who are non-preeclamptic or arecarrying chromosomally normal fetuses. This selected group shouldcomprise a sufficient number of women such that the average level offetal/placental derived RNA transcribed from genetic loci (which may becoding for a particular fetal protein or may be non-coding) reflects,with reasonable accuracy, the level of RNA in the general population ofhealthy pregnant women with healthy fetuses. In addition, the selectedgroup of women should have a similar gestational age to that of a womanwhose blood is tested for indication of preeclampsia or fetalchromosomal aneuploidy such as trisomy 18 and trisomy 21. The preferredgestational age for practicing the present invention may vary, dependingon the disorder that is being screened for. For example, a pregnantwoman is screened for the risk of preeclampsia preferably during thesecond trimester of the pregnancy, whereas fetal chromosomal aneuploidyis preferably screened for and diagnosed as early as possible. Moreover,the preferred gestational age for testing may also depend on the RNAmarker used in testing, as certain markers may be more readilydetectable during some stages of gestation than in others stages.

The term “average” may be used similarly to refer to the amount ofspecified RNA species that is representative of the amount found in theblood of a randomly selected group of healthy non-pregnant women.

IGFBP3, ABP1, FN1, SLC21A2, KIAA0992, TIMP3, LPL, INHBA, LEP, SIGLEC6,RPL17, COL6A1, COL6A2, SOD1, APP, BTG3, ATP5J, ADAMTS1, BACE2, DSCR5,ITSN1, PLAC4, LOC90625, ATP5O, EFEMP1, and TFRC, as used herein, referto the genes or proposed open reading frames (including their variantsand mutants) and their polynucleotide transcripts as exemplified by thesequences set forth in GenBank Accession Nos. provided in Tables 2, 4,and 6. In some context, these terms may also be used to refer to thepolypeptides encoded by these genes or open reading frames.

“Standard control” as used herein refers to a sample suitable for theuse of a method of the present invention, in order for quantitativelydetermining the amount of RNA transcript, e.g., COL6A1, COL6A2, APP,ATP5O, or LEP. Such a sample contains a known amount of thefetal/placental derived RNA species that closely reflects the averagelevel of such RNA in an average pregnant woman. Similarly, a “standardcontrol” may be derived from an average healthy non-pregnant woman.

“An increase or a decrease in the amount of mRNA from the standardcontrol” as used herein refers to a positive or negative change inamount from the standard control. An increase is preferably at least2-fold, more preferably at least 5-fold, and most preferably at least10-fold. Similarly, a decrease is preferably at least 50%, morepreferably at least 80%, and most preferably at least 90%.

A “polynucleotide hybridization method” as used herein refers to amethod for detecting the presence and/or quantity of a polynucleotidebased on its ability to form Watson-Crick base-pairing, underappropriate hybridization conditions, with a polynucleotide probe of aknown sequence. Examples of such hybridization methods include Southernblotting and Northern blotting.

“PCR primers” as used herein refer to oligonucleotides that can be usedin a polymerase chain reaction (PCR) to amplify a nucleotide sequenceoriginated from an RNA transcript derived from a genetic locus, such asCOL6A1, COL6A2, APP, ATP5O, or LEP. At least one of the PCR primers foramplification of an RNA sequence derived from an above-named locusshould be sequence-specific for the said locus.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention provides, for the first time, methods and kits fordiagnosing, monitoring, or predicting preeclampsia and fetal chromosomalaneuploidy such as trisomy 18 and trisomy 21 in pregnant women, as wellas for detecting pregnancy in women, by analyzing the level of one ormore of several fetal/placental derived RNA species, i.e., those withsequences set forth in Tables 1-6, present in the women's blood.

According to the invention, the amount of these RNA transcripts offetal/placental origin in a maternal blood sample can be quantitativelydetermined, preferably following an amplification procedure, e.g.,reverse transcriptase polymerase chain reaction (RT-PCR). The amount ofone or more of these RNA species is then compared to a standard controlhaving an RNA level of the same species that is representative of anaverage pregnant woman without these pregnancy-related disorders at asimilar gestational age. An increase or decrease in the RNA levelindicates the presence of or an increased risk of developing thedisorders. The present invention thus provides a novel approach fordiagnosis of preeclampsia and fetal chromosomal aneuploidy such astrisomy 18 and trisomy 21, which is non-invasive as well as gender- andpolymorphism-independent.

Relying on the same methodology, by comparing the level of one or moreof the RNA species transcribed from these genetic loci in a woman'sblood to an established control value obtained from average non-pregnantwoman, the present invention may be used to detect pregnancy.

Although fetal/placental expressed RNA has been used as markers forprenatal diagnosis and monitoring, see, e.g., U.S. patent applicationSer. Nos. 09/876,005 and 10/759,783, the identification of anyparticular RNA species as a suitable marker for this purpose is afinding of unpredictable nature, as not all species of RNA expressed inplacenta can be detected in maternal blood. For instance, the presentinventors have been unable to detect certain fetal/placental derived RNAspecies in the maternal blood. Some exemplary species that areundetectable include: NADH dehydrogenase (ubiquitnone) flavoprotein 3,10 kDa (NDUFV3); alpha-fetoprotein (AFP); hemoglobin, epsilon 1 (HBE1);and phospholipase A2, group IIA (platelets, synovila fluid) (PLA2G2A).

II. Preparation of Blood Samples

A. Obtaining Blood Samples

The first step of practicing the present invention is to obtain abiological sample, e.g., a blood sample, from a pregnant woman at agestational age suitable for testing using a method of the presentinvention, or from a woman who is being tested for possible pregnancy.The suitable gestational age may vary depending on the disorder testedand sometimes the RNA marker used, as discussed above. Collection ofblood from a woman is performed in accordance with the standard protocolhospitals or clinics generally follow. An appropriate amount ofperipheral blood, e.g., between 3-20 ml, is collected and maybe storedaccording to standard procedure prior to further preparation.

B. Preparing Plasma or Serum Samples

The serum or plasma of a woman's blood is suitable for the presentinvention and can be obtained by well known methods. For example, awoman's blood can be placed in a tube containing EDTA or a specializedcommercial product such as Vacutainer SST (Becton Dickinson, FranklinLakes, N.J.) to prevent blood clotting, and plasma can then be obtainedfrom whole blood through centrifugation. On the other hand, serum isobtained through centrifugation following blood clotting. Centrifugationis typically conducted at an appropriate speed, e.g., 1,500-3,000×g, ina chilled environment, e.g., at a temperature of about 4-10° C. Plasmaor serum may be subject to additional centrifugation steps before beingtransferred to a fresh tube for RNA extraction. In certain applicationsof this invention, plasma or serum may be the preferred sample types. Inother applications of the present invention, whole blood may bepreferable. Yet in other applications, other fractions of blood may bepreferable.

III. Quantitative Determination of the Amount of RNA in a Woman's Blood

A. Extraction of RNA

There are numerous methods for extracting RNA from a biological sample.The general methods of RNA preparation (e.g., described by Sambrook andRussell, Molecular Cloning: A Laboratory Manual 3d ed., 2001) can befollowed; various commercially available reagents or kits, such asTrizol reagent (Invitrogen, Carlsbad, Calif.), Oligotex Direct mRNA Kits(Qiagen, Valencia, Calif.), RNeasy Mini Kits (Qiagen, Hilden, Germany),and PolyATtract® Series 9600™ (Promega, Madison, Wis.), may also be usedto obtain RNA from a blood sample from a woman. Combinations of morethan one of these methods may also be used.

It is preferable in some applications that all or most of thecontaminating DNA be eliminated from the RNA preparations. Thus, carefulhandling of the samples, thorough treatment with DNase, and propernegative controls in the amplification and quantification steps shouldbe used.

B. PCR-Based Quantitative Determination of RNA Level

Once RNA is extracted from a woman's blood sample, the amount of RNAderived from a genetic locus of interest, e.g., COL6A1, COL6A2, APP,ATP5O, or LEP, may be quantified. The preferred method for determiningthe RNA level is an amplification-based method, e.g., by PCR.

Prior to the amplification step, a DNA copy (cDNA) of the RNA ofinterest must be synthesized. This is achieved by reverse transcription,which can be carried out as a separate step, or in a homogeneous reversetranscription-polymerase chain reaction (RT-PCR), a modification of thepolymerase chain reaction for amplifying RNA. Methods suitable for PCRamplification of ribonucleic acids are described by Romero and Rotbartin Diagnostic Molecular Biology: Principles and Applications pp.401-406; Persing et al., eds., Mayo Foundation, Rochester, Minn., 1993;Egger et al., J. Clin. Microbiol. 33:1442-1447, 1995; and U.S. Pat. No.5,075,212.

The general methods of PCR are well known in the art and are thus notdescribed in detail herein. For a review of PCR methods, protocols, andprinciples in designing primers, see, e.g., Innis, et al., PCRProtocols: A Guide to Methods and Applications, Academic Press, Inc.N.Y., 1990. PCR reagents and protocols are also available fromcommercial vendors, such as Roche Molecular Systems.

PCR is most usually carried out as an automated process with athermostable enzyme. In this process, the temperature of the reactionmixture is typically cycled through a denaturing region, a primerannealing region, and an extension reaction region automatically. Insome protocols, the annealing region and the extension reaction regionare merged. Machines specifically adapted for this purpose arecommercially available.

Although PCR amplification of the target RNA is typically used inpracticing the present invention. One of skill in the art willrecognize, however, that amplification of these RNA species in amaternal blood sample may be accomplished by any known method, such asligase chain reaction (LCR), transcription-mediated amplification, andself-sustained sequence replication or nucleic acid sequence-basedamplification (NASBA), each of which provides sufficient amplification.More recently developed branched-DNA technology may also be used toquantitatively determining the amount of RNA markers in maternal blood.For a review of branched-DNA signal amplification for directquantification of nucleic acid sequences in clinical samples, see Nolte,Adv. Clin. Chem. 33:201-235, 1998.

C. Other Quantitative Methods

The RNA species of interest can also be detected using other standardtechniques, well known to those of skill in the art. Although thedetection step is typically preceded by an amplification step,amplification is not required in the methods of the invention. Forinstance, the RNA species of interest may be identified by sizefractionation (e.g., gel electrophoresis), whether or not preceded orfollowed by an amplification step. After running a sample in an agaroseor polyacrylamide gel and labeling with ethidium bromide according towell known techniques (see, e.g., Sambrook and Russell, supra), thepresence of a band of the same size as the standard control is anindication of the presence of a target RNA, the amount of which may thenbe compared to the control based on the intensity of the band.Alternatively, oligonucleotide probes specific to RNA transcribed from agenetic locus, e.g., COL6A1, COL6A2, APP, ATP5O, or LEP, can be used todetect the presence of such RNA species and indicate the amount of RNAin comparison to the standard control, based on the intensity of signalimparted by the probe.

Sequence-specific probe hybridization is a well known method ofdetecting a particular nucleic acid comprising other species of nucleicacids. Under sufficiently stringent hybridization conditions, the probeshybridize specifically only to substantially complementary sequences.The stringency of the hybridization conditions can be relaxed totolerate varying amounts of sequence mismatch.

A number of hybridization formats well known in the art, including butnot limited to, solution phase, solid phase, or mixed phasehybridization assays. The following articles provide an overview of thevarious hybridization assay formats: Singer et al., Biotechniques 4:230,1986; Haase et al., Methods in Virology, pp. 189-226, 1984; Wilkinson,In situ Hybridization, Wilkinson ed., IRL Press, Oxford UniversityPress, Oxford; and Hames and Higgins eds., Nucleic Acid Hybridization: APractical Approach, IRL Press, 1987.

The hybridization complexes are detected according to well knowntechniques and the detection is not a critical aspect of the presentinvention. Nucleic acid probes capable of specifically hybridizing to atarget nucleic acid, i.e., the RNA species of interest or the amplifiedDNA, can be labeled by any one of several methods typically used todetect the presence of hybridized nucleic acids. One common method ofdetection is the use of autoradiography using probes labeled with ³H,¹²⁵I, ³⁵S, ¹⁴C, or ³²P, or the like. The choice of radioactive isotopedepends on research preferences due to ease of synthesis, stability, andhalf lives of the selected isotopes. Other labels include compounds(e.g., biotin and digoxigenin), which bind to antiligands or antibodieslabeled with fluorophores, chemiluminescent agents, and enzymes.Alternatively, probes can be conjugated directly with labels such asfluorophores, chemiluminescent agents or enzymes. The choice of labeldepends on the sensitivity required, ease of conjugation with the probe,stability requirements, and available instrumentation.

The probes and primers necessary for practicing the present inventioncan be synthesized and labeled using well known techniques.Oligonucleotides used as probes and primers may be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage and Caruthers, Tetrahedron Letts.,22:1859-1862, 1981, using an automated synthesizer, as described inNeedham-VanDevanter et al., Nucleic Acids Res. 12:6159-6168, 1984.Purification of oligonucleotides is by either native acrylamide gelelectrophoresis or by anion-exchange high performance liquidchromatography (HPLC) as described in Pearson and Regnier, J. Chrom.,255:137-149, 1983.

IV. Establishing a Standard Control

In order to establish a standard control, a group of healthy pregnantwomen carrying healthy fetuses should first be selected. These womenshould be of similar gestational age, which is within the appropriatetime period of pregnancy for screening of conditions such aspreeclampsia and fetal chromosomal aneuploidies (including trisomy 18 ortrisomy 21) using the methods of the present invention. Similarly, astandard control is established using samples from a group of healthynon-pregnant women.

The health status of the selected pregnant women and the fetuses theyare carrying should be confirmed by well established, routinely employedmethods including but not limited to monitoring blood pressure of thewomen, recording the onset of labor, and conducting fetal geneticanalysis using CVS and amniocentesis.

Furthermore, the selected group of healthy pregnant women carryinghealthy fetuses or healthy non-pregnant women must be of a reasonablesize, such that the average amount of RNA derived from the genetic locinamed in this application calculated from the group can be reasonablyregarded as representative of the normal or average amount among thegeneral population of healthy women carrying healthy fetuses or healthynon-pregnant women. Preferably, the selected group comprises at least 10women.

Once an average value is established for the amount of fetal/placentalderived RNA based on the individual values found in each women of theselected group, this value is considered a standard for the RNA species.Any blood sample that contains a similar amount of RNA of the samespecies can thus be used as a standard control. A solution containingRNA species of interest with a concentration of the established averageof the same species can also be artificially assembled and serve as astandard control.

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially similar results.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially the same or similar results.

Example 1 Genetic Loci on Chromosome 21 or 18 with Expression inPlacental Tissues

Methods

Subjects

Placental tissue and blood samples were collected with informed consentfrom pregnant women during the first trimester, who attended theDepartment of Obstetrics and Gynecology at the Prince of Wales Hospital,Hong Kong. The study was approved by the Clinical Research EthicsCommittee.

Sample Preparation for Microarray Analysis

Five first-trimester placental tissue samples were obtained frompregnant women by chorionic villus sampling (CVS) before therapeuticterminations. Fetal karyotypes in all cases were subsequently confirmedto be normal. The placental tissue samples were stored in RNAlater™(Ambion®, Austin, Tex.) immediately upon collection and kept at −80° C.until RNA extraction. Six milliliters of maternal peripheral blood werecollected concurrently at the time of tissue collection and stored inPAXgene™ Blood RNA Tubes (PreAnalytiX, Hombrechtikon, Switzerland).Total RNA from placental tissues were extracted with Trizol Reagent(Invitrogen, Carlsbad, Calif.) and purified with the RNeasy mini-kit(Qiagen, Hilden, Germany) following manufacturers' protocols. Total RNAfrom peripheral blood was extracted by the PAXgene™ Blood RNA Kit(PreAnalytiX, Hombrechtikon, Switzerland) according to manufacturer'sinstructions, with the inclusion of DNase treatment (RNase-Free DNaseSet, Qiagen, Hilden, Germany).

Gene Expression Analysis by High-Density Oligonucleotide Microarrays

For each sample, ten micrograms of the extracted RNA were labeled andhybridized to the GeneChip® Human Genome U133A and U133B Arrays(Affymetrix, Santa Clara, Calif.) according to the manufacturer'sinstructions. After hybridization, each array was washed and stained ina GeneChip® Fluidics Station 400 (Affymetrix, Santa Clara, Calif.). Thechips were scanned with the GeneArray Scanner (Affymetrix, Santa Clara,Calif.) and analyzed using the GeneChip® Microarray Suite 5.0(Affymetrix).

Real-Time Quantitative RT-PCR

One-step real-time quantitative RT-PCR (QRT-PCR) was used for thequantitative measurement of RNA transcripts in placental tissues andmaternal blood samples. QRT-PCR assays for the detection of thehouse-keeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH)have been described previously (Ng et al. 2002). Sequences of theprimers (Proligo, Singapore) and fluorescent probes (Applied Biosystems,Foster City, Calif., USA) of the other studied genes are shown in Table1A. For placental tissues and maternal buffy coat analyses, relativequantification was employed wherein the studied transcript levels werenormalized to the corresponding GAPDH mRNA levels.

The QRT-PCR reactions were set up according to the manufacturer'sinstructions (EZ rTth RNA PCR reagent set, Applied Biosystems) in areaction volume of 25 μl. The QRT-PCR assays were carried out in acombined thermal cycler and fluorescent detector (ABI Prism 7900HT,Applied Biosystems). For all transcripts, the PCR primers and thefluorescent probes were used at concentrations of 300 nM and 100 nM,respectively. Before performing QRT-PCR, contaminating DNA in theplacental tissue RNA extracts was removed by DNase I digestion(Invitrogen, Carlsbad, Calif.) according to the manufacturer'srecommendations. 17 ng of extracted placental RNA was used foramplification. Multiple negative water blanks were included in everyanalysis.

The thermal profiles used were as follows: the reaction was initiated at50° C. for 2 min for the included uracil N-glycosylase to act, followedby reverse transcription at 60° C. for 30 min. After a 5-mindenaturation at 95° C., 40 cycles of PCR were carried out usingdenaturation at 92° C. for 15 s and 1 min annealing/extension at 58° C.

Quantitative Assessment of Placental Expressed Transcripts in MaternalBlood

Maternal whole blood samples from normal pregnant women were collectedinto EDTA tubes. After centrifugation of the blood samples at 1,600 gfor 10 min at 4° C., the buffy coat and plasma fractions were carefullytransferred into separate polypropylene tubes. The plasma samples werere-centrifuged at 16,000 g for 10 min at 4° C. Supernatants werecollected into fresh polypropylene tubes. RNA extraction from theharvested maternal plasma was performed as previously described (Ng etal., 2002). RNA was similarly extracted from 0.3 mL of the buffy coatfraction.

QRT-PCR assays for the studied transcripts were carried out with thesame conditions described above. Five microliters of the extractedplasma RNA or 10 ng of the buffy coat RNA were used for each QRT-PCRreaction. Absolute quantification was used to determine the transcriptconcentrations in plasma samples. Calibration curves were prepared byserial dilutions of high performance liquid chromatography-purifiedsingle stranded synthetic DNA oligonucleotides (Proligo, Singapore)spanning the full lengths of the amplicons, with concentrations rangingfrom 1×10⁷ copies to 1×10¹ copies. Absolute concentrations of thetranscripts in plasma were expressed as copies/ml of plasma. Thesequences of the synthetic DNA oligonucleotides are shown in Table 1B.Results for the buffy coat fractions were expressed by relativequantification based on normalization to GAPDH.

Statistical Analysis

Statistical analysis was performed using the Sigma Stat 2.03 software(SPSS).

Results

Identification of Placental Expressed Genes by High-DensityOligonucleotide Microarrays

Gene expression profiles of five first-trimester CVS samples wereobtained by independent microarray analysis of each individual tissuesample. Among the ˜22,000 well-characterized transcripts detectable bythe Human Genome U133A and U133B Arrays (Affymetrix), a total of 7226gene transcripts were expressed in the CVS samples. We have previouslyreported that circulating DNA in the plasma of normal individuals ispredominantly derived from hematopoietic cells (Lui et al., 2002). Thus,we hypothesize that much of the background maternal nucleic acids inmaternal blood also originate from the hematopoietic compartment. As weaim to identify placenta-expressed transcripts amongst the circulatingRNA molecules in maternal plasma, we further obtained the geneexpression profiles of maternal whole blood and compared these profileswith those of the corresponding placental tissues using the GeneChip®Microarray Suite 5.0 software (Affymetrix). Placental expressedtranscripts in early pregnancy were identified by selecting transcriptswhose expression levels were “increased” in the CVS tissues whencompared to the corresponding whole blood samples in all five sets ofcomparisons. After this procedure, transcripts that were expressed inmaternal blood cells to a higher or similar degree as that of placentaltissues were eliminated. Thus, this analysis has resulted in theidentification of a panel of 1245 transcripts with relative placentalspecificity in the first trimester of pregnancy.

Selection of Genetic Loci Encoded on Chromosome 21 or 18 with Expressionin Placental Tissues

Among the panel of transcripts with relative placental specificity asidentified by the approach described above, we further sought fortranscripts that were derived from genes positioned on chromosome 21.Thirteen genes that are located on chromosome 21 have been identifiedand are summarized in Table 2A. This gene selection strategy is based onthe reasoning that the altered gene dosage as a result of the presenceof an additional chromosome 21 in the genome of a fetus with trisomy 21,may lead to aberrant expression of genes located on chromosome 21. As wehave previously shown that the placenta is an important source ofcirculating fetal RNA in maternal plasma (Ng et al., 2003), aberrantplacental tissue expression of the targeted genes as a result of trisomy21 may be reflected by aberrant concentrations of the said transcriptsin maternal blood. Thus, one approach for the noninvasive prenataldetection of fetal trisomy 21 through circulating fetal/placentalderived RNA analysis is based on the detection of abnormal bloodconcentrations of those selected transcripts in women with fetusesaffected by trisomy 21 in comparison to that in women conceived with anormal fetus.

A similar strategy had been applied for the identification of genemarkers potentially useful for the noninvasive prenatal assessment oftrisomy 18. The gene expression profiles of both the CVS and maternalwhole blood samples were analyzed by the Human Genome U133B Arrays(Affymetrix). A panel of transcripts with preferential expression in CVSwith respect to maternal blood was identified with the use of the samescreening criteria described above. Placental expressed genes which arelocated on chromosome 18 were selected from the panel of transcriptswith relative placental specificity. Within the panel, the transcriptwith the highest expression level in CVS was selected and shown in Table2B.

Validation of Microarray Results by Real-Time QRT-PCR

Placental tissue expression of the markers identified from themicroarray-based strategy described above were verified by one-stepreal-time QRT-PCR. First trimester CVS tissues from ten normalpregnancies and three trisomy 21 pregnancies were measured for GAPDHmRNA and the selected transcripts listed in Tables 2A and 2B. Therelative mRNA levels of the studied genes were normalized to thecorresponding GAPDH levels using the equation:ΔCt _(X) =Ct _(GAPDH) −Ct _(X)where Ct represents the threshold cycle which is the number of PCRcycles required for the accumulated fluorescence of the QRT-PCR reactionof a sample to reach a predetermined threshold intensity. ΔCt_(X) is thenormalized mRNA level of a studied transcript, X; Ct_(GAPDH) is the Ctvalue of GAPDH mRNA; and Ct_(X) is the Ct value of the transcript, X. Asthe Ct value is inversely proportional to the logarithm of the amount oftemplate mRNA, greater ΔCt_(X) values represent higher mRNA levels. Thestudied transcripts have been confirmed to be expressed and detectablein the CVS tissues collected from normal as well as pregnanciesinvolving a trisomy 21 fetus.

Statistically significant up-regulations in the placental tissueexpression of ADAMTS1 mRNA (FIG. 1A) (Mann-Whitney test, P=0.036) andAPP mRNA (FIG. 1B) (Mann-Whitney test, P=0.036) were found in the CVStissues collected from trisomy 21 pregnancies in comparison to normalpregnancies. These data confirmed our hypothesis that genes located onthe trisomic chromosome are associated with quantitative aberrations inplacental tissue expression and thus, are potentially useful markers forthe prenatal assessment of trisomy 21.

Detectability of the Placental Expressed Transcripts in Maternal Blood

Detectability of some of the transcripts was assessed in buffy coat andplasma samples collected from women in the third trimester of pregnancy.All of the twelve studied transcripts were detectable in both the buffycoat (FIG. 2) and plasma samples (data not shown). To test for thepregnancy specificity of the transcripts, plasma samples from tenpregnant women before delivery and at 24 hours after delivery were alsocollected. FIGS. 3A and 3B reveal that both COL6A1 and COL6A2 mRNA werepromptly cleared from the maternal plasma after delivery (Wilcoxon test,P<0.05 for both cases), while the corresponding plasma GAPDH mRNA levelsremained unchanged (data not shown, Wilcoxon test, P=1.000). Thepost-delivery clearance of COL6A1 and COL6A2 mRNA from maternal plasmasuggests that the placenta is the predominant tissue source of thesetranscripts.

Conclusion

Using a microarray-based approach, transcripts that are expressed infirst trimester placental tissues were identified. Thirteen transcriptsthat are useful for the prenatal assessment of trisomy 21 wereidentified based on the selection of placental expressed genes that arelocated on chromosome 21. Similarly, an RNA marker that is useful forthe prenatal assessment of trisomy 18 was identified through theselection of placental transcripts that are encoded on chromosome 18.

The detectability of the studied transcripts in both normal andaneuploid placental tissues were confirmed by real-time QRT-PCR. Asexamples, ADAMTS1 and APP mRNA were shown to be aberrantly expressed inplacental tissues of trisomy 21 pregnancies. In addition, mRNA of allthe targeted genes were found to be detectable in maternal buffy coatand plasma. These data confirm that our marker selection strategyenables the identification of RNA species that are aberrantly expressedin trisomy 21 placental tissues and are detectable in maternalcirculation which would facilitate the development of strategies for thenoninvasive prenatal diagnosis of fetal trisomy 21. For example,noninvasive prenatal assessment of trisomy 21 could be based on thedetection of the aberrant concentrations of the RNA markers in maternalblood of trisomy 21 pregnancies in comparison to those of normalpregnancies. Alternatively, noninvasive prenatal diagnosis could becarried out based on the relative quantitative comparison of differentmolecular forms of one or more of the transcripts in maternal plasma.Similar applications can also be applied to trisomy 18 with thedetection of RPL17 mRNA in maternal plasma.

Example 2 Genes with Increased Expression in Placentas of Trisomy 21Pregnancies Compared with that of Normal Pregnancies

Methods

Subjects

All placental tissue and blood samples in this study were collected withinformed consent from women in the first trimester of pregnancy, whoattended the Department of Obstetrics and Gynecology at the Prince ofWales Hospital, Hong Kong. The study was approved by the ClinicalResearch Ethics Committee.

In the first part of the study, placental tissue gene expressionprofiles of both the normal and trisomy 21 pregnancies were identifiedby oligonucleotide microarray. First-trimester placental tissue sampleswere obtained from pregnant women by chorionic villus sampling (CVS).Five women with normal pregnancies (gestational age range: 10-12 weeks)and three pregnant women conceived with trisomy 21 fetuses (gestationalage range: 12-13 weeks) were recruited with the respective fetalkaryotype subsequently confirmed. In the second part of the study, thegene expression profiles generated by the oligonucleotide microarrayexperiments were confirmed using QRT-PCR. CVS from three trisomy 21pregnancies (gestational age range: 13-14 weeks) and 5 normal pregnantwomen (gestational age range: 9-13 weeks) were recruited for this partof the study.

Sample Preparation for Microarray Analysis

CVS samples were stored in RNAlater™ (Ambion®, Austin, Tex.) immediatelyupon collection and kept at −80° C. until RNA extraction. For the fivepregnant women with normal pregnancies, six milliliters of maternalperipheral blood were collected concurrently at the time of tissuecollection and stored in PAXgene™ Blood RNA Tubes (PreAnalytiX,Hombrechtikon, Switzerland). Total RNA from placental tissues wereextracted with Trizol Reagent (Invitrogen, Carlsbad, Calif.) andpurified with the RNeasy mini-kit (Qiagen, Hilden, Germany) followingmanufacturers' protocols. Total RNA from peripheral blood was extractedby the PAXgene™ Blood RNA Kit (PreAnalytiX, Hombrechtikon, Switzerland)according to manufacturer's instructions, with the inclusion of DNasetreatment (RNase-Free DNase Set, Qiagen, Hilden, Germany).

Gene Expression Analysis by High-Density Oligonucleotide Microarrays

For each sample, ten micrograms of the extracted RNA were labeled andhybridized to the GeneChip® Human Genome U133A and U133B Arrays(Affymetrix, Santa Clara, Calif.) according to the manufacturer'sinstructions. After hybridization, each array was washed and stained ina GeneChip® Fluidics Station 400 (Affymetrix, Santa Clara, Calif.). Thechips were scanned with the GeneArray Scanner (Affymetrix, Santa Clara,Calif.) and analyzed using the GeneChip® Microarray Suite 5.0(Affymetrix).

Real-Time Quantitative RT-PCR

One-step real-time QRT-PCR was used for the quantitative measurement ofmRNA transcripts in placental tissues and maternal plasma samples.QRT-PCR assays for the detection of the house-keeping gene, GAPDH havebeen described previously (Ng et al., 2002). Sequences of the primers(Proligo, Singapore) and TaqMan minor-groove-binding (MGB) fluorescentprobes (Applied Biosystems, Foster City, Calif., USA) of the otherstudied genes are shown in Table 3. The mRNA quantities were expressedusing relative quantifications wherein the studied transcript levelswere normalized to the corresponding GAPDH mRNA levels.

The QRT-PCR reactions were set up according to the manufacturer'sinstructions (EZ rTth RNA PCR reagent set, Applied Biosystems) in areaction volume of 25 μl. The QRT-PCR assays were carried out in acombined thermal cycler and fluorescent detector (ABI Prism 7900HT,Applied Biosystems). For all transcripts, the PCR primers and thefluorescent probes were used at concentrations of 300 nM and 100 nM,respectively. Before performing QRT-PCR, contaminating DNA in theextracted placental tissue RNA was removed by DNase I digestion(Invitrogen, Carlsbad, Calif.) according to the manufacturer'srecommendations. 17 ng of placental RNA extracts was used foramplification. Multiple negative water blanks were included in everyanalysis.

The thermal profiles used for all of the studied transcripts were asfollows: the reaction was initiated at 50° C. for 2 min for the includeduracil N-glycosylase to act, followed by reverse transcription at 60° C.for 30 min. After a 5-min denaturation at 95° C., 40 cycles of PCR werecarried out using denaturation at 92° C. for 15 s and 1 minannealing/extension at 58° C.

Quantitative Assessment of Trisomy 21 Associated Placental Transcriptsin Maternal Blood

Maternal whole blood samples from pregnant women were collected intoEDTA tubes. After centrifugation of the blood samples at 1,600 g for 10min at 4° C., plasma was carefully transferred into plain polypropylenetubes. The plasma samples were re-centrifuged at 16,000 g for 10 min at4° C. Supernatants were collected into fresh polypropylene tubes. RNAextraction from the harvested maternal plasma was performed aspreviously described (Ng et al., 2002). QRT-PCR assays for the studiedtranscripts were carried out with conditions described above. Fivemicroliters of the extracted plasma RNA were used for each QRT-PCRreaction.

Statistical Analysis

Statistical analysis was performed using the Sigma Stat 2.03 software(SPSS).

Results

Microarray-Based Identification of Genes with Aberrant Placental TissueExpression in Aneuploid Pregnancies

Gene expression profiles of the five first-trimester CVS samplescollected from normal pregnancies were obtained by independentmicroarray analysis of each individual tissue sample. We have previouslyreported that circulating DNA in the plasma of normal individuals ispredominantly derived from hematopoietic cells (Lui et al., 2002). Thus,we hypothesize that much of the background maternal nucleic acids inmaternal blood also originate from the hematopoietic compartment. As theultimate aim of the study was to identify placental expressedtranscripts that are fetal specific amongst the circulating RNAmolecules in maternal blood, we further obtained the gene expressionprofiles of paired maternal whole blood and compared these profiles withthose of the corresponding CVS for the five normal pregnancy samples.GeneChip® Microarray Suite 5.0 software (Affymetrix) was used for thecomparison. Transcripts with relative placental specificity wereidentified by selecting transcripts whose expression levels were‘increased’ in the CVS tissues when compared to the corresponding wholeblood samples in all five sets of comparisons. After these procedures,transcripts that were expressed in maternal blood cells to a higher orsimilar degree to that of CVS tissues were eliminated. This procedurehas resulted in the identification of a panel of transcripts which arepreferentially expressed in placental tissues.

In the next step, transcripts that are aberrantly expressed in placentaltissues of aneuploid pregnancies were identified. Using the GeneChip®Microarray Suite 5.0 software (Affymetrix), expression profiles of threetrisomy 21 CVS tissues were compared with the panel of genes withrelative placental specificity identified from five gestational-agematched normal pregnancies as described above. Gene expression signalsof the three aneuploid CVS samples were compared individually with thatof each of the five normal CVS samples using the normal placental tissueexpression profiles as baselines. A total of 15 comparisons wereperformed and the number of comparisons which showed up-regulatedexpression in the aneuploid placentas were counted (I-count) for each ofthe genes interrogated. The fold-changes in expression levels werecalculated and were converted to log₂ values (Signal Log Ratio, SLR).Transcripts were further selected if: (i) the transcripts wereup-regulated in aneuploid placentas when compared to normal placentas tothe extent where the Signal Log Ratio of at least 0.4 (1.3-fold changein expression); and (ii) the up-regulations were consistent where morethan half of the comparisons revealed such up-regulations (I-count≧8).Table 4 summarizes the microarray results of three transcripts, namelyEGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1),transferrin receptor p90 CD71 (TFRC), and ATP5O, which arepreferentially expressed in placentas with the greatest extent ofup-regulations among the gene panels for trisomy 21 pregnancies.

Validation of Microarray Results by Real-Time QRT-PCR

The three transcripts with aberrant placental tissue expression intrisomy 21 pregnancies as identified from the microarray experimentsdescribed above were verified by one-step real-time QRT-PCR. mRNA levelsof the three transcripts and GAPDH were quantified in CVS tissuescollected from three trisomy 21 and five normal pregnancies matched forgestational age. The relative mRNA levels of the studied genes werenormalized to the corresponding GAPDH levels using the equation:ΔCt _(X) =Ct _(GAPDH) −Ct _(X)where Ct represents the threshold cycle which is the number of PCRcycles required for the accumulated fluorescence of the QRT-PCR reactionof a sample to reach a predetermined threshold intensity. ΔCt_(X) is thenormalized mRNA level of a studied transcript, X; Ct_(GAPDH) is the Ctvalue of GAPDH mRNA; and Ct_(X) is the Ct value of the transcript, X. Asthe Ct value is inversely proportional to the logarithm of the amount oftemplate mRNA, greater ΔCt_(X) values represent higher mRNA levels.

The QRT-PCR analysis revealed that EFEMP1 mRNA (FIG. 4A), TFRC mRNA(FIG. 4B) and ATP5O mRNA (FIG. 4C) were indeed up-regulated in thetrisomy 21 CVS when compared to CVS collected from normal pregnancies.The aberrant placental tissue expression of the three transcripts ispresent in aneuploid pregnancies, thus demonstrates their utility as RNAmarkers for the prenatal investigation of trisomy 21.

Detectability of the RNA Markers in Maternal Plasma

Plasma samples from normal pregnant women were measured for ATP5O mRNA.ATP5O mRNA is detectable in maternal plasma (FIG. 4D) with astatistically significant decrease in its concentration 24 hours afterdelivery (FIG. 4D; Wilcoxon, P<0.05). These data indicate that theplacenta is an important tissue source of ATP5O mRNA in maternal plasma.

Conclusion

Using a microarray-based approach, transcripts with aberrant expressionsin trisomy 21 placental tissues were identified. The three transcripts,EFEMP1, TFRC, and ATP5O, were identified by the microarray experimentsand the aberrant nature of their expression in placental tissues oftrisomy 21 pregnancies were further verified by QRT-PCR. These data thusindicate that the three transcripts are useful as RNA markers for theprenatal assessment of fetal trisomy 21. The detectability of ATP5O mRNAin maternal plasma indicates the suitability of this transcript for thenoninvasive prenatal assessment of fetal trisomy 21. For example,noninvasive prenatal assessment of trisomy 21 could be based on thedetection of the aberrant concentrations of the RNA markers in maternalplasma of trisomy 21 pregnancies in comparison to that of normalpregnancies.

Example 3 Genes with Aberrant Expression in Placentas of PregnanciesAffected by Preeclampsia Compared with that of Normal Pregnancies

Methods

Subjects

All placental tissue and blood samples in this study were collected withinformed consent from women in the third trimester of pregnancy, whoattended the Department of Obstetrics and Gynecology at the Prince ofWales Hospital, Hong Kong. The study was approved by the ClinicalResearch Ethics Committee.

In the first part of the study, placental tissue gene expressionprofiles of both normal and preeclamptic (PET) pregnancies wereidentified by oligonucleotide microarray. Placental tissues from 5 PETpregnant women (gestational age range: 37-40 weeks) and 5 healthypregnant women (gestational age range: 38-40 weeks) were obtainedimmediately after cesarean section. Peripheral blood was collectedimmediately before delivery. In the second part of the study, the geneexpression profiles generated from the oligonucleotide microarrayexperiments were confirmed using QRT-PCR. Placentas from 10 PET(gestational age range: 25-40 weeks) and 10 healthy pregnant women(gestational age range: 37-39 weeks) were collected immediately aftercesarean delivery. Preeclampsia was defined on the basis of a sustainedincrease in diastolic blood pressure >110 mm Hg on one occasion or >90mm Hg on two or more occasions at least 4 hours apart, with the presenceof significant proteinuria in women with no history of hypertension.Significant proteinuria was defined as proteinuria >0.3 g/day or ≧2+ ondipstick testing in two clean-catch midstream urine specimens collectedat least 4 hours apart.

Sample Preparation for Microarray Analysis

Placental tissue samples were stored in RNAlater™ (Ambion®, Austin,Tex.) immediately upon collection and kept at −80° C. until RNAextraction. Six milliliters of maternal peripheral blood were collectedconcurrently at the time of tissue collection and stored in PAXgene™Blood RNA Tubes (PreAnalytiX, Hombrechtikon, Switzerland). Total RNAfrom placental tissues were extracted with Trizol Reagent (Invitrogen,Carlsbad, Calif.) and purified with the RNeasy mini-kit (Qiagen, Hilden,Germany) following manufacturers' protocols. Total RNA from peripheralblood was extracted by the PAXgene™ Blood RNA Kit (PreAnalytiX,Hombrechtikon, Switzerland) according to manufacturer's instructions,with the inclusion of DNase treatment (RNase-Free DNase Set, Qiagen,Hilden, Germany).

Gene Expression Analysis by High Density Oligonucleotide Microarrays

For each sample, ten micrograms of the extracted RNA were labeled andhybridized to the GeneChip® Human Genome U133A and U133B Arrays(Affymetrix, Santa Clara, Calif.) according to the manufacturer'sinstructions. After hybridization, each array was washed and stained ina GeneChip® Fluidics Station 400 (Affymetrix, Santa Clara, Calif.). Thechips were scanned with the GeneArray Scanner (Affymetrix, Santa Clara,Calif.) and analyzed using the GeneChip® Microarray Suite 5.0(Affymetrix).

Real-Time Quantitative RT-PCR

One-step real-time QRT-PCR was used for the quantitative measurement ofmRNA transcripts in placental tissues and maternal plasma samples.QRT-PCR assays for the detection of the house-keeping gene, GAPDH havebeen described previously (Ng et al., 2002). Sequences of the primersand TaqMan minor-groove-binding (MGB) fluorescent probes (AppliedBiosystems, Foster City, Calif., USA) of the other studied genes areshown in Table 5. The mRNA quantities were expressed using relativequantifications wherein the studied transcript levels were normalized tothe corresponding GAPDH mRNA levels.

The QRT-PCR reactions were set up according to the manufacturer'sinstructions (EZ rTth RNA PCR reagent set, Applied Biosystems) in areaction volume of 25 μl. The QRT-PCR assays were carried out in acombined thermal cycler and fluorescent detector (ABI Prism 7900HT,Applied Biosystems). For all of the studied transcripts, the PCR primers(Proligo) and the fluorescent probes (Applied Biosystems) were used atconcentrations of 300 nM and 100 nM, respectively. Before performingQRT-PCR, contaminating DNA in the placental tissue RNA extracts wasremoved by DNase I digestion (Invitrogen, Carlsbad, Calif.) according tothe manufacturer's recommendations. 17 ng of extracted placental RNA wasused for amplification. Multiple negative water blanks were included inevery analysis.

The thermal profiles used were as follows: the reaction was initiated at50° C. for 2 min for the included uracil N-glycosylase to act, followedby reverse transcription at 60° C. for 30 min. After a 5-mindenaturation at 95° C., 40 cycles of PCR were carried out usingdenaturation at 92° C. for 15 s and 1 min annealing/extension at 56° C.

Quantitative Assessment of Preeclampsia-Associated Placental Transcriptsin Maternal Blood

Maternal whole blood samples from pregnant women were collected intoEDTA tubes. After centrifugation of the blood samples at 1,600 g for 10min at 4° C., plasma was carefully transferred into plain polypropylenetubes. The plasma samples were re-centrifuged at 16,000 g for 10 min at4° C. Supernatants were collected into fresh polypropylene tubes. RNAextraction from the harvested maternal plasma was performed aspreviously described (Ng et al., 2002). QRT-PCR assays for the studiedtranscripts were carried out with conditions described above. Fivemicroliters of the extracted plasma RNA were used for each QRT-PCRreaction.

Statistical Analysis

Statistical analysis was performed using the Sigma Stat 2.03 software(SPSS).

Results

Microarray-Based Identification of Genes with Aberrant Placental TissueExpression in Preeclamptic Pregnancies

Our ultimate goal is to develop an approach for the investigation of PETthrough the detection of PET-associated transcripts in maternal blood.Therefore, our gene selection strategy would first entail theidentification of transcripts which are preferentially expressed in thePET placentas but not in the maternal peripheral blood cells. Thisstrategy was devised based on our previous finding that the placenta isan important source of circulating fetal RNA in maternal blood and thehematopoietic system is the main source of plasma DNA in normalindividuals (Lui et al., 2002). Gene expression profiles of 5 PETplacental tissue samples and their corresponding peripheral bloodsamples were determined by oligonucleotide microarray. To identifyplacental expressed genes, transcripts which were expressed in at least4 of the 5 analyzed PET placental tissue samples were selected. Genesthat were also expressed in maternal blood cells were then eliminatedthrough the positive selection of transcripts whose expression levelswere either “absent” in all of the 5 peripheral blood samples or“increased” in the placentas when compared to the corresponding wholeblood samples in all of the five sets of paired placentas and maternalblood samples. Thus, transcripts that were expressed in a similar orgreater extent in the maternal blood cells than the placental tissueswere eliminated. These procedures resulted in the selection of a panelof transcripts which were preferentially expressed in placental tissues.

In the next step, transcripts with aberrant expression in PET placentaswere identified. Expression profiles of placentas collected from 5 eachof PET and normal pregnancies matched for gestational ages were comparedusing GeneChip® Microarray Suite 5.0 software (Affymetrix). Expressionsignals of the list of relatively placental specific genes identifiedfrom the 5 PET pregnancies as described above were compared individuallywith that of each of the 5 normal placental tissue samples using thenormal placental tissue expression profiles as baselines. A total of 25comparisons were performed and the number of comparisons which showedup-regulated expression in the PET placentas were counted (I-count) foreach of the genes interrogated. The fold-changes in expression levelswere calculated and were converted to log₂ values (Signal Log Ratio,SLR). Transcripts were further selected if: (i) the transcripts wereup-regulated in PET placentas when compared to normal placentas to theextent where the Signal Log Ratio of at least 0.4 (1.3-fold change inexpression) and (ii) the up-regulations were consistent where more thanhalf of the comparisons revealed such up-regulations (I-count≧13). Table6 summarizes the microarray results of the ten identified transcriptswhich are preferentially expressed in placentas with up-regulations inPET pregnancies.

Validation of Microarray Results by Real-Time QRT-PCR

The PET-related transcripts selected from the microarray analyses wereverified by one-step real-time QRT-PCR. GAPDH mRNA concentrations weremeasured in placental tissues collected from ten each of PET and normalpregnancies matched for gestational age. The GAPDH mRNA levels were usedto normalize the transcript levels between different samples. Theexpression levels of the ten transcripts identified by the microarrayanalyses were then assessed in the placental tissue samples of bothgroups of pregnancies. The relative mRNA levels of the studied geneswere normalized to the corresponding GAPDH levels using the equation:ΔCt _(X) =Ct _(GAPDH) −Ct _(X)where Ct represents the threshold cycle which is the number of PCRcycles required for the accumulated fluorescence of the QRT-PCR reactionof a sample to reach a predetermined threshold intensity. ΔCt_(X) is thenormalized mRNA level of a studied transcript, X; Ct_(GAPDH) is the Ctvalue of GAPDH mRNA; and Ct_(X) is the Ct value of the transcript, X. Asthe Ct value is inversely proportional to the logarithm of the amount oftemplate mRNA, greater ΔCt_(X) values represent higher mRNA levels.

Leptin (LEP) and sialic acid binding Ig-like lectin 6 (SIGLEC6) mRNAwere confirmed to be significantly up-regulated in PET placentas whencompared with those of normal pregnancies by the QRT-PCR analyses (FIGS.5A, and 5B for Leptin, and SIGLEC6 mRNA, respectively) (Mann-Whitneytest, P<0.05 for both cases). These data confirm that our transcriptselection strategy enables the identification of markers that areaberrantly expressed in PET placental tissues.

Detectability of the PET-Related RNA Markers in Maternal Plasma

Plasma samples from 25 healthy and 26 PET-affected pregnancies in thethird trimester were measured for LEP and INHBA mRNA by QRT-PCR.Maternal plasma concentrations for LEP and INHBA were significantlyelevated in pregnancies affected by PET when compared with theuncomplicated pregnancies (LEP: FIG. 6A, Mann-Whitney, P=0.017; INHBA:FIG. 6B, Mann Whitney, P=0.006).

Conclusion

Using a microarray-based approach, transcripts with differentialexpression in PET placentas were identified and were considered aspotential markers for the investigation of PET. Within the list ofPET-associated transcripts identified by the micorarray analyses, tentranscripts that are most aberrantly expressed in PET placentas comparedto that of normal pregnancies were selected. Real-time QRT-PCR confirmedthat both LEP and SIGLEC6 expressions were significantly up-regulated inPET placentas when compared with normal placentas. Maternal plasmalevels of INHBA and LEP were significantly higher in PET thanuncomplicated pregnancies and thus suggest the possibility of the use ofthe markers for the noninvasive prenatal assessment of PET.

Example 4 Placental-Specific PLAC4 mRNA in Maternal Plasma of Trisomy 21and Normal Pregnancies

Determination of Detectability and Pregnancy-Specificity of PLAC4 mRNA

The PLAC4 mRNA can be detected in maternal plasma using real-timeQRT-PCR assays. In addition, the PLAC4 mRNA was cleared from thematernal plasma following the birth of the child. Thus, the PLAC4 mRNAin maternal plasma is of fetal origin and is pregnancy-specific.

Sample Collection and Processing

Peripheral blood samples from five non-pregnant women, fivefirst-trimester and eight third-trimester pregnant women were collected.Peripheral blood from six third-trimester pregnant women before and at24 hours after delivery was also obtained. The blood samples werecollected in EDTA tubes. Plasma samples were harvested as described inExample 1. RNA extraction from maternal plasma samples was performedfollowing the procedures described in Example 1.

Development of Real-Time QRT-PCR Assay

The QRT-PCR assay for PLAC4 mRNA was developed as described inExample 1. The sequences of the primers (Integrated DNA Technologies,Coralville, Iowa), TaqMan minor groove binding (MGB) fluorescent probes(Applied Biosystems, Foster City, Calif., USA) and the calibrator(Proligo, Singapore) are shown in Table 7.

The QRT-PCR reactions were set up according to the manufacturer'sinstructions (EZ rTth RNA PCR reagent set, Applied Biosystems) in areaction volume of 25 μl. The QRT-PCR assays were carried out in an ABIPRISM® 7900HT (Applied Biosystems, Foster City, Calif., USA). The PCRprimers and the fluorescent probe were used at concentrations of 400 nMand 100 nM, respectively. 5 μl of extracted RNA were used foramplification. The thermal cycling profile was: the reaction wasinitiated at 50° C. for 2 min, followed by reverse transcription at 60°C. for 30 min. After a 5-min denaturation at 95° C., 45 cycles of PCRwere carried out using denaturation at 95° C. for 15 s and 1 min at 60°C.

PLAC4 mRNA can be Detected in Maternal Plasma and are Pregnancy-Specific

The PLAC4 mRNA could be detected in none of the non-pregnant individualsbut all of the first- and third-trimester pregnant women (FIG. 7). Themedian plasma PLAC4 mRNA concentrations in the first- andthird-trimester pregnancies were 299.6 copies/ml and 529.3 copies/ml,respectively. The pregnancy-specificity of the circulating PLAC4 mRNAwas also determined. In the pre-delivery plasma samples, the medianPLAC4 mRNA concentration was 500.0 copies/ml. The transcript wasundetectable in any of the postpartum plasma samples (FIG. 8).

Comparison of Circulating PLAC4 mRNA in Euploid and Trisomy 21Pregnancies

Circulating PLAC4 mRNA concentrations were compared betweenkaryotypically normal and trisomy 21 pregnancies. Plasma samples werecollected from 29 pregnant women carrying euploid fetuses and fivepregnant women carrying trisomy 21 fetuses during the first- andsecond-trimester of pregnancy. The plasma samples were measured forPLAC4 mRNA concentrations by real-time one-step RT-PCR as described.PLAC4 mRNA was detected in all of the trisomic plasma samples. Themedians for the trisomy 21 and normal pregnancies are 5581 copies/ml and4836 copies/ml, respectively. Due to the small sample size, nostatistically significant difference was established for the plasmaPLAC4 mRNA concentrations between the normal and the trisomy 21pregnancies.

Example 5 Genes with Aberrant Expression in Placentas of PregnanciesAffected by Preeclampsia Compared with that of Normal Pregnancies

Methods

Subjects

All placental tissue and blood samples in this study were collected withinformed consent from women in the third trimester of pregnancy, whoattended the Department of Obstetrics and Gynaecology at the Prince ofWales Hospital, Hong Kong. The study was approved by the ClinicalResearch Ethics Committee.

In the first part of the study, placental tissue gene expressionprofiles of both normal and preeclamptic (PET) pregnancies wereidentified by oligonucleotide microarray. Placental tissues from 5 PETpregnant women (gestational age range: 37-40 weeks) and 5 healthypregnant women (gestational age range: 38-40 weeks) were obtainedimmediately after cesarean section. Peripheral blood was collectedimmediately before delivery. In the second part of the study, the geneexpression profiles generated from the oligonucleotide microarrayexperiments were confirmed using QRT-PCR. Placentas from 6 PET(gestational age range: 30-39 weeks) and 6 healthy pregnant women(gestational age range: 37-39 weeks) were collected immediately aftercesarean delivery. Preeclampsia was defined on the basis of a sustainedincrease in diastolic blood pressure >110 mm Hg on one occasion or >90mm Hg on two or more occasions at least 4 hours apart, with the presenceof significant proteinuria in women with no history of hypertension.Significant proteinuria was defined as proteinuria >0.3 g/day or ≧2+ ondipstick testing in two clean-catch midstream urine specimens collectedat least 4 hours apart.

Sample Preparation for Microarray Analysis

Placental tissue samples were stored in RNAlater™ (Ambion®, Austin,Tex.) immediately upon collection and kept at −80° C. until RNAextraction. Six milliliters of maternal peripheral blood were collectedconcurrently at the time of tissue collection and stored in PAXgene™Blood RNA Tubes (PreAnalytiX, Hombrechtikon, Switzerland). Total RNAfrom placental tissues were extracted with Trizol Reagent (Invitrogen,Carlsbad, Calif.) and purified with the RNeasy mini-kit (Qiagen, Hilden,Germany) following manufacturers' protocols. Total RNA from peripheralblood was extracted by the PAXgene™ Blood RNA Kit (PreAnalytiX,Hombrechtikon, Switzerland) according to manufacturer's instructions,with the inclusion of DNase treatment (RNase-Free DNase Set, Qiagen,Hilden, Germany).

Gene Expression Analysis by High Density Oligonucleotide Microarrays

For each sample, ten micrograms of the extracted RNA were labeled andhybridized to the GeneChip® Human Genome U133A and U133B Arrays(Affymetrix, Santa Clara, Calif.) according to the manufacturer'sinstructions. After hybridization, each array was washed and stained ina GeneChip® Fluidics Station 400 (Affymetrix, Santa Clara, Calif.). Thechips were scanned with the GeneArray Scanner (Affymetrix, Santa Clara,Calif.) and analyzed using GeneSpring v 7.2 (Agilent Technologies, PaloAlto, Calif.).

Mining of Microarray Gene Expression Data

The microarray data were imported into GeneSpring v 7.2 (AgilentTechnologies) in the .CEL format. Data mining was performedindependently for samples (placental tissues and maternal blood cells)collected from the normal and PET pregnancies. Within each group ofpregnancies, genes that had relatively higher expression in theplacental tissue samples than the maternal blood cells were firstidentified. Microarray raw data from the 5 placentas and paired maternalblood cells were normalized together using the following steps insequence: (1) raw data processing by Robust Multi-chip Average, withGC-content background correction (GC-RMA); (2) data transformationwhereby microarray data with values below 0.001 were set to 0.001; and(3) the signal intensity for each gene was divided by the median of itsmeasurements in all samples. Genes with statistically significant(P<0.05) expression in either the placental tissues or maternal bloodcells were further identified. These genes were then sorted in the orderbased on the fold-differences in the placental tissue expression incomparison to that of the maternal blood cells with the aim ofidentifying transcripts with high fold-differences. This data miningprocess would lead to the identification of genes with relatively higherexpression in placental tissues compared with maternal blood cells.

On the other hand, data mining was performed to identify genes with highabsolute expression levels in placental tissues. The raw microarray datafor the placental tissues were normalized by GC-RMA processing followedby data transformation whereby microarray data with values below 0.001were set to 0.001. Genes were then ranked based on the normalizedexpression levels. Data mining for the placental tissues collected fromthe normal and preeclamptic pregnancies were performed independently.

Genes were selected for further investigation if they demonstrated muchhigher fold-differences between the PET placentas in relation to thepaired maternal blood than that for the normal pregnancies, or thosewith much higher absolute expression levels in the PET than the normalplacentas while demonstrating at least 200-fold difference between theplacental tissue and maternal blood expression.

Real-Time Quantitative RT-PCR

One-step real-time QRT-PCR was used for the quantitative measurement ofmRNA transcripts in placental tissues. A calibration curve was preparedby serial dilution of a high performance liquid chromatography-purifiedsingle stranded synthetic DNA oligonucleotide with concentrationsranging from 2.5×10⁶ copies to 2.5 copies. Sequences of the primers(Proligo), fluorescent probes (Applied Biosystems, Foster City, Calif.,USA) and oligonucleotide calibrators of the studied genes are shown inTable 8.

The QRT-PCR reactions were set up according to the manufacturer'sinstructions (EZ rTth RNA PCR reagent set, Applied Biosystems) in areaction volume of 50 μl. The QRT-PCR assays were carried out in acombined thermal cycler and fluorescent detector (ABI Prism 7900HT,Applied Biosystems). For all of the studied transcripts, the fluorescentprobes were used at concentrations of 100 nM. 300 nM each of the forwardand reverse primers were used for each reaction in the assays forpregnancy-associated plasma protein A, pappalysin 1 (PAPPA), INHBA andFN1. 400 nM each of the forward and reverse primers were used for eachreaction in the assays for LEP, ADAM metallopeptidase domain 12 (meltrinalpha) (ADAM12), and pappalysin 2 (PAPPA2). Before performing QRT-PCR,contaminating DNA in the placental tissue RNA extracts was removed byDNase I digestion (Invitrogen, Carlsbad, Calif.) according to themanufacturer's recommendations. 1 ng of extracted placental RNA was usedfor amplification. Multiple negative water blanks were included in everyanalysis. Placental tissue RNA concentrations were expressed ascopies/ng of placental total RNA.

The thermal profiles used were as follows: the reaction was initiated at50° C. for 2 min for the included uracil N-glycosylase to act, followedby reverse transcription at 60° C. for 30 min. After a 5-mindenaturation at 95° C., 40 cycles of PCR were carried out usingdenaturation at 92° C. for 15 s and 1 min annealing/extension at 56° C.for LEP, ADAM12, PAPPA and INHBA, but at 57° C. for PAPPA2 and FN1.

Statistical Analysis

Statistical analysis was performed using the Sigma Stat 3.0 software(SPSS).

Results

Genes that were identified from the microarray analysis included LEP,ADAM12 (GenBank Accession No. NM_(—)003474, NM_(—)021641), PAPPA(GenBank Accession No. NM_(—)002581), PAPPA2 (GenBank Accession No.NM_(—)020318, NM_(—)021936), INHBA and FN1. Placental tissue expressionlevels of the selected transcripts in PET and normal pregnancies wereassessed by one-step real-time QRT-PCR. The results are shown in FIG. 9.The concentrations for LEP, ADAM12, PAPPA2, INHBA and FN1 were found tobe higher in placental tissues collected from PET than normalpregnancies, while that for PAPPA mRNA was found to be lower inplacental tissues collected from PET than normal pregnancies.

Conclusion

Using a microarray-based approach, transcripts with aberrant expressionprofiles in PET placentas were identified and were considered aspotential markers for the investigation of PET. Six transcripts wereselected from the microarray analyses and the aberrant nature of theirexpression profile in PET placentas is confirmed by real-time QRT-PCR.

REFERENCES

-   Lui, Y Y N, Chik, K W, Chiu, R W K, Ho, C Y, Lam, C W and Lo, Y M D    (2002). Predominant hematopoietic origin of cell-free DNA in plasma    and serum after sex-mismatched bone marrow transplantation. Clin    Chem 48, 421-427.-   Ng, E K O, Tsui, N B Y, Lam, N Y, Chiu, R W K, Yu, S C, Wong, S C,    Lo, E S, Rainer, T H, Johnson, P J and Lo, Y M D (2002). Presence of    filterable and nonfilterable mRNA in the plasma of cancer patients    and healthy individuals. Clin Chem 48, 1212-1217.-   Ng, E K O, Tsui, N B Y, Lau, T K, Leung, T N, Chiu, R W K, Panesar,    N S, Lit, L C W, Chan, K W and Lo, Y M D (2003). mRNA of placental    origin is readily detectable in maternal plasma. Proc Natl Acad Sci    U.S.A. 100, 4748-4753.

All patents, patent applications, and other publications cited in thisapplication, including published amino acid or polynucleotide sequences,are incorporated by reference in the entirety for all purposes. TABLE 1ASequences of primers and probes for real-time QRT-PCR detection of theplacental expressed transcripts encoded on chromosome 21. TranscriptSequence COL6A1 F primer GACAAAGTCAAGTCCTTCACCAA Probe(FAM)CGCTTCATCGACAACC(MGBNFQ) R primer GCGTTCCACACCAGGTTT COL6A2 Fprimer GATCAACCAGGACACCATCAA Probe (FAM)CGCATCATCAAGGTC(MGBNFQ) R primerCCGTAGGCTTCGTGTTTCA SOD1 F primer CAGGGCATCATCAATTTCG Probe(FAM)CAGAAGGAAAGTAATGGACCA(MGBNFQ) R primer TGCTTCCCCACACCTTCA ATP5O Fprimer CCCTCACTACCAACCTGATCA Probe (FAM)TGCTTGCTGAAAATG(MGBNFQ) R primerCCTTGGGTATTGCTTAATCGA BTG3 F primer GATGTCCTGAAAGCCTGTGAA Probe(FAM)ACAGCTGCATCTTGT(MGBNFQ) R primer GGCAAGCCCAGGTCACTA APP F primerAAGGAAGGCATCCTGCAGTA Probe (FAM)TGCCAAGAAGTCTACC(MGBNFQ) R primerACATTGGTGATCTGCAGTTCA ATP5J F primer CCTGTCCGAATCAGCATGAT Probe(FAM)CTTCAGAGGCTCTTCA(MGBNFQ) R primer TGACCGAATGACAGAGGAGAA ADAMTS1 Fprimer CCACAGGAACTGGAAGCATAA Probe (FAM)AAAGAAGCGATTTGTGTCCA(MGBNFQ) Rprimer CAAGCATGGTTTCCACATAGC BACE2 F primer GGAATGGAATACTTGGCCTAGCTProbe (FAM)ATGCCACACTTGCCAAGCCATCAAGTT(TA MRA) R primerCACCAGGGAGTCGAAGAAGGT DSCR5 F primer GAATCTTGGCTAAACTCTTTAGGTTT Probe(FAM)ACCTATTGGCCTCAAAAA(MGBNFQ) R primer AGGTAATGCAACTGCCCAAT ITSN1 Fprimer TGGTGGCAGCCTGGATA Probe (FAM)CTGGGCCATAACTG(MGBNFQ) R primerATCATGCTTCGCTCTTTCCT PLAC4 F primer CCTTTCCCCCTTATCCAACT Probe(FAM)CCCTAGCCTATACCC(MGBNFQ) R primer GTACTGGTTGGGCTCATTTTCT L0C90625 Fprimer TGCACATCGGTCACTGATCT Probe (FAM)CCTACTGGCACAGACG(MGFNFQ) R primerGGTCAGTTTGGCCGATAAAC RPL17 F primer TGAGGGTTGACTGGATTGGT Probe(FAM)AGGCCCGTGTGGCT(MGBNFQ) R primer TACAGCACTGCTTCCACAGAAMGBNFQ: minor-groove-binding non-fluorescent quencher; FAM: fluorescentreporter; TAMRA: fluorescent quencher.

TABLE 1B Sequences of the oligonucleotide calibrators used in theabsolute quantification of the placental expressed transcripts encodedon chromosome 21. Transcripts Calibrator Sequence COL6A1TGGACAAAGTCAAGTCCTTCACCAAGCGCTTCATCGACAACCTGAGGGACAGGTACTACCGCTGTGACCGAAACCTGGTGTGGAACGCAG COL6A2GAGATCAACCAGGACACCATCAACCGCATCATCAAGGTCATGAAACACGAAGCCTACGGAG ATP5OTCCCCTCACTACCAACCTGATCAATTTGCTTGCTGAAAATGGTCGATTAAGCAATACCCAAGGAG SOD1TGCAGGGCATCATCAATTTCGAGCAGAAGGAAAGTAATGGACCAGTGAAGGTGTGGGGAAGCATT

TABLE 2A Microarray detection of placental expressed genes located onChromosome 21. GenBank *Signal Probe Set ID accession no. TranscriptsSymbol Location (median) 213428_s_at AA292373 Collagen, type VI, alpha 1COL6A1 21q22.3 8419.2 200642_at NM_000454.1 superoxide dismutase 1,soluble (amyotrophic SOD1 21q22.11 7084.7 lateral sclerosis 1 (adult))209156_s_at AY029208.1 Collagen, type VI, alpha 2 COL6A2 21q22.3 7076.9200818_at NM_001697.1 ATP synthase, H+ transporting, mitochondrial F1ATP5O 21q22.11 3247.8 complex, O subunit (oligomycin sensitivityconferring protein) 213134_x_at AI765445 BTG family, member 3 BTG321q21.1 2564.9 214953_s_at X06989.1 amyloid beta (A4) precursor protein(protease nexin- APP 21q21.3 2376.1 II, Alzheimer disease) 202325_s_atNM_001685.1 ATP synthase, H+ transporting, mitochondrial F0 ATP5J21q21.1 2303.1 complex, subunit F6 214750_at L13197 placenta-specific 4PLAC4 21q22.3 2209.9 222162_s_at AK023795.1 a disintegrin-like andmetalloprotease (reprolysin ADAMTS1 21q21.2 1780.8 type) withthrombospondin type 1 motif, 1 217867_x_at NM_012105.1 beta-siteAPP-cleaving enzyme 2 BACE2 21q22.3 1093.4 221689_s_at AB035745.1 Downsyndrome critical region gene 5 DSCR5 21q22.2 900.7 209298_s_atAF114488.1 intersectin 1 (SH3 domain protein) ITSN1 21q22.1-q22.2 199.9^(#)232191_at BC005107.1 hypothetical protein BC005107 LOC90625 21q22.36910.2*Medians of microarray signals from five first trimester placentaltissues^(#)Transcripts that were detected by Human Genome U133B Arrays(Affymetrix). Transcripts without specification were detected by HumanGenome U133A Arrays (Affymetrix)

TABLE 2B Transcript with the highest expression level in first-trimesterplacentas among the placental expressed genes located on Chromosome 18.The gene was detected by Human Genome U133B Arrays (Affymetrix). GenBank*Signal Probe Set ID accession no. Transcripts Symbol Location (median)200038_s_at NM_000985.1 ribosomal protein RPL17 Chr: 18q21 25603.6 L17*Medians of microarray signals from five first trimester placentaltissues

TABLE 3 Sequences of primers and probes for real-time QRT-PCR detectionof the placental expressed transcripts with aberrant expression intrisomy 21. Transcript Sequence TFRC F primer CGGCTGCAGGTTCTTCTG Probe(FAM)TGGCAGTTCAGAATGA(MGBNFQ) R primer GTTAGAGAATGCTGATCTAGCTTGA EFEMP1F primer CACAACGTGTGCCAAGACAT Probe (FAM)ACGCACAACTGTAGAGCA(MGBNFQ) Rprimer CGTAAATTGATGCACACTTGGT ATP5O F primer CCCTCACTACCAACCTGATCA Probe(FAM)TGCTTGCTGAAAATG(MGBNFQ) R primer CCTTGGGTATTGCTTAATCGAMGBNFQ: minor-groove-binding non-fluorescent quencher

TABLE 4 Microarray detection of placental expressed genes withdifferential expression between trisomy 21 and normal CVS tissues. Thegenes were detected by Human Genome U133A Arrays (Affymetrix) GenBank*Signals Probe Set ID accession no. Transcript Symbol (Median) I-count201842_s_at AI826799 EGF-containing fibulin-like extracellular matrixEFEMP1 11244 11 protein 1 207332_s_at NM_003234 transferrin receptor(p90, CD71) TFRC 10645.8 11 ATP synthase, H+ transporting, mitochondrial200818_at NM_001697 F1 complex, O subunit (oligomycin sensitivity ATP5O5516.1 15 conferring protein)*Medians of microarray signals from 3 trisomy 21 CVS

TABLE 5 Sequences of primers and probes for real-time QRT-PCR detectionof the preeclampsia-associated placental expressed transcripts.Transcript Sequence IGFBP3 F primer AGTCCAAGCGGGAGACAG Probe(FAM)AATATGGTCCCTGCCG(MGBNFQ) R primer CAGGTGATTCAGTGTGTCTTCC ABP1 Fprimer TGGAAGCAGAGCGAACTG Probe (FAM)AGCGAGAGATGCC(MGBNFQ) R primerCATCAGGATGGCAGCCA FN1 F primer AAGCAAGCCCGGTTGTTA Probe(FAM)ACACTATCAGATAAATCAAC(MGBNFQ) R primer CCAACGCATTGCCTAGGTA INHBA Fprimer CGCCCTCCCAAAGGAT Probe (FAM)TACCCAACTCTCAGCCAGAGATGGTG(TAMRA) Rprimer GCATGTTTAAAATGTGCTTCTTG SLC21A2 F primer GCTTTGGGCTCTCCAGTTCProbe (FAM)TTTCCAGCTTGAATGAGA(MGBNFQ) R primer GTAGCTGACAAAGATGATGAGGATSIGLEC6 F primer CAAGCTCTCTGTGCGTG Probe (FAM)ATGGCCCTGACCCA(MGBNFQ) Rprimer GTCCCTGGGATGGAGATGT KIAA0992 F primer ACCTGTTTGGCTACGAATCC Probe(FAM)ACATCTGCTGAGGTGTT(MGBNFQ) R primer GAATCTGTTGAACTGGCACCTT TIMP3 Fprimer CCTTCTGCAACTCCGACAT Probe (FAM)CGTGATCCGGGCCA(MGBNFQ) R primerAGCTTCTTCCCCACCACC LEP F primer GGTGAGAGCTGCTCTGGAAA Probe(FAM)TGACCCAGATCCTC(MGBNFQ) R primer CCTCAGCCTGATTAGGTGGTT LPL F primerAGCAAAACCTTCATGGTGATC Probe (FAM)TGGCTGGACGGTAAC(MGBNFQ) R primerGCACCCAACTCTCATACATTCCMGBNFQ: minor-groove-binding non-fluorescent quencher; FAM: fluorescentreporter; TAMRA: fluorescent quencher.

TABLE 6 Microarray detection of placental-expressed genes withdifferential expression between preeclamptic and normal placentaltissues. The genes were detected by Human Genome U133A and U133B Arrays(Affymetrix) GenBank *PET Signals ^(#)SLR Probe Set ID accession no.Transcript Symbol (Median) I-count (Median) 210095_s_at M31159insulin-like growth factor binding protein 3 IGFBP3 16136.5 16 0.5203559_s_at NM_001091 amiloride binding protein 1 (amine oxidase ABP113574.5 19 1.4 (copper-containing) 210495_x_at AF130095 fibronectin 1FN1 13005.7 13 0.4 210511_s_at M13436 inhibin, beta A (activin A,activin AB alpha INHBA 10425.5 13 0.7 polypeptide) 204368_at NM_005630solute carrier family 21 (prostaglandin transporter), SLC21A2 3800.9 150.6 member 2 210796_x_at D86359 sialic acid binding Ig-like lectin 6SIGLEC6 3731.5 16 0.8 200897_s_at NM_016081 palladin KIAA0992 3098.5 130.4 201150_s_at NM_000362 tissue inhibitor of metalloproteinase 3(Sorsby TIMP3 2979.4 13 0.4 fundus dystrophy, pseudoinflammatory)207092_at NM_000230 leptin (obesity homolog, mouse) LEP 2056.6 13 0.8203549_s_at NM_000237 lipoprotein lipase LPL 1727.0 13 0.5*Medians of microarray signals from five preeclamptic placental tissues^(#)SLR denotes signal log ratio

TABLE 7 Sequences of the PCR primers, the probe and the calibrator forreal-time QRT-PCR detection of PLAC4 mRNA. Primer Sequence (5′ to 3′) Fprimer CCTTTCCCCCTTATCCAACT R primer GTACTGGTTGGGCTCATTTTCT Probe(FAM)CCCTAGCCTATACCC(MGBNFQ) CalibratorCACCTTTCCCCCTTATCCAACTAGCCCTAGCCTATACCCTCTGCTGCCCAAGAAAATGAGCCCAACCAGTACACMGBNFQ: minor groove binding non-fluorescent quencher

TABLE 8 Sequences of primers, probes and calibrators for real-timeQRT-PCR detection of the preeclampsia-associated placental expressedtranscripts. Transcript Sequence FN1 F primer AAGCAAGCCCGGTTGTTA Probe(FAM)ACACTATCAGATAAATCAAC(MGBNFQ) R primer CCAACGCATTGCCTAGGTACalibrator AAAGCAAGCCCGGTTGTTATGACAATGGAAAACACTATCAGATAAATCAACAGTGGGAGCGGACCTACCTAGGCAATGCGTTGGT INHBA F primer CGCCCTCCCAAAGGATProbe (FAM)TACCCAACTCTCAGCCAGAGATGGTG(TAMRA) R primerGCATGTTTAAAATGTGCTTCTTG CalibratorCCGCCCTCCCAAAGGATGTACCCAACTCTCAGCCAGAGATGGTGGAGGCCGTCAAGAAGCACATTTTAAACATGCT LEP F primer GGTGAGAGCTGCTCTGGAAA Probe(FAM)TGACCCAGATCCTC(MGBNFQ) R primer CCTCAGCCTGATTAGGTGGTT CalibratorGGGTGAGAGCTGCTCTGGAAAATGTGACCCAGATCCTCACAACCACCTAATCAGGCTGAGGT ADAM12 Fprimer TGGAAAGAAATGAAGGTCTCATTG Probe (FAM)CACGGAAACCCACTATCTGCAAGACGGTA(TAMRA) R primer TCGAGCGAGGGAGACATCA CalibratorTGGAAAGAAATGAAGGTCTCATTGCCAGCAGTTTCACGGAAACCCACTATCTGCAAGACGGTACTGATGTCTCCCTCGCTCGAA PAPPA2 F primer CACAGTGGAAGCCTGGGTTAA Probe(FAM)CCGGAGGGAGGACAGAACAACCCA(TAMRA) R primer ATCAAACACACCTGCGATGATGCalibratorTCACAGTGGAAGCCTGGGTTAAACCGGAGGGAGGACAGAACAACCCAGCCATCATCGCAGGTGTGTTTGATA PAPPA F primer GGGCATTCACACCATCAGT ProbeFAM-CCAAGACAACAAAGACCCACGCTACT-TAMRA R primer TCGGTCTGTCTTCAAGGAGAACalibratorTGGGCATTCACACCATCAGTGACCAAGACAACAAAGACCCACGCTACTTTTTCTCCTTGAAGACAGACCGAGMGBNFQ: minor-groove-binding non-fluorescent quencher; FAM: fluorescentreporter; TAMRA: fluorescent quencher.

1. A method for diagnosing, monitoring, or predicting preeclampsia in apregnant woman, the method comprising the steps of: (i) quantitativelydetermining the amount of one or more RNA species in a biological sampleobtained from the pregnant woman, wherein the RNA species areindependently selected from RNA derived from genetic loci consisting ofIGFBP3, ABP1, FN1, SLC21A2, KIAA0992, TIMP3, LPL, INHBA, LEP, ADAM12,PAPPA, PAPPA2, and SIGLEC6, and wherein the biological sample is blood,washing from the reproductive tract, amniotic fluid, urine, saliva, orchorionic villus; and (ii) comparing the amount of the RNA species fromstep (i) to a standard control representing the amount of the RNAspecies in the corresponding sample from an average non-preeclampticpregnant woman, wherein an increase or a decrease in the amount of theRNA species from the standard control indicates preeclampsia or anincreased risk of developing preeclampsia.
 2. The method of claim 1,wherein the RNA species is derived from ADAM12, PAPPA2, FN1, INHBA, LEP,or SIGLEC6, and wherein an increase in the amount of the RNA speciesfrom the standard control indicates preeclampsia or an increased risk ofdeveloping preeclampsia.
 3. The method of claim 1, wherein the RNAspecies is derived from PAPPA and wherein a decrease in the amount ofthe RNA species from the standard control indicates preeclampsia or anincreased risk of developing preeclampsia.
 4. The method of claim 1,wherein step (i) comprises using a reverse transcriptase polymerasechain reaction (RT-PCR).
 5. The method of claim 4, wherein step (i)further comprises using mass spectrometry following RT-PCR.
 6. Themethod of claim 1, wherein step (i) comprises using a polynucleotidehybridization method.
 7. The method of claim 1, wherein step (i)comprises using a primer extension reaction.
 8. The method of claim 1,wherein the woman is during the first trimester of gestation.
 9. Themethod of claim 1, wherein the woman is during the second or thirdtrimester of gestation.
 10. The method of claim 1, wherein the blood isplasma.
 11. The method of claim 1, wherein the blood is serum.
 12. Themethod of claim 1, wherein the increase in the amount of RNA from thestandard control is more than 2-fold.
 13. The method of claim 1, whereinthe decrease in the amount of RNA from the standard control is more than50%.
 14. A kit for diagnosing, monitoring, or predicting preeclampsia ina pregnant woman, the kit comprising: (i) PCR primers for quantitativelydetermining the amount of one or more RNA species in a biological sampleobtained from the pregnant woman, wherein the RNA species isindependently selected from RNA derived from genetic loci consisting ofIGFBP3, ABP1, FN1, SLC21A2, KIAA0992, TIMP3, LPL, INHBA, LEP, ADAM12,PAPPA, PAPPA2, and SIGLEC6, and wherein the biological sample is blood,washing from the reproductive tract, amniotic fluid, urine, saliva, orchorionic villus; and (ii) a standard control representing the amount ofthe RNA species in the corresponding sample from an averagenon-preeclamptic pregnant woman.
 15. A method for detecting the presenceof a fetus with trisomy 18 in a pregnant woman, the method comprisingthe steps of: (i) quantitatively determining the amount of an RNAspecies in a biological sample obtained from the pregnant woman, whereinthe RNA species is derived from RPL17, and wherein the biological sampleis blood, washing from the reproductive tract, amniotic fluid, urine,saliva, or chorionic villus; and (ii) comparing the amount of the RNAspecies from step (i) to a standard control representing the amount ofthe RNA species in the corresponding sample from an average pregnantwoman with a chromosomally normal fetus, wherein an increase in theamount of the RNA species from the standard control indicates anincreased risk of having a fetus with trisomy
 18. 16. The method ofclaim 15, wherein step (i) comprises using reverse transcriptasepolymerase chain reaction (RT-PCR).
 17. The method of claim 16, whereinstep (i) further comprises using mass spectrometry.
 18. The method ofclaim 15, wherein step (i) comprises using a polynucleotidehybridization method.
 19. The method of claim 15, wherein step (i)comprises using a primer extension reaction.
 20. The method of claim 15,wherein the woman is during the first trimester of gestation.
 21. Themethod of claim 15, wherein the woman is during the second or thirdtrimester of gestation.
 22. The method of claim 15, wherein the blood isplasma.
 23. The method of claim 15, wherein the blood is serum.
 24. Themethod of claim 15, wherein the increase in the amount of mRNA from thestandard control is more than 2-fold.
 25. The method of claim 15,wherein the decrease in the amount of mRNA from the standard control ismore than 50%.
 26. A kit for detecting the presence of a fetus withtrisomy 18 in a pregnant woman, the kit comprising: (i) PCR primers forquantitatively determining the amount of an RNA species in a biologicalsample obtained from the pregnant woman, wherein the RNA species isderived from RPL17, and wherein the biological sample is blood, washingfrom the reproductive tract, amniotic fluid, urine, saliva, or chorionicvillus; and (ii) a standard control representing the amount of the RNAspecies in a corresponding sample from an average pregnant woman with achromosomally normal fetus.
 27. A method for detecting the presence of afetus with trisomy 21 in a pregnant woman, the method comprising thesteps of: (i) quantitatively determining the amount of one or more RNAspecies in a biological sample obtained from the pregnant woman, whereinthe RNA species is independently selected from RNA species derived fromgenetic loci consisting of COL6A1, COL6A2, SOD1, APP, BTG3, ATP5J,ADAMTS1, BACE2, DSCR5, ITSN1, PLAC4, ATP5O, LOC90625, EFEMP1, and TFRC,and wherein the biological sample is blood, washing from thereproductive tract, amniotic fluid, urine, saliva, or chorionic villus;and (ii) comparing the amount of the RNA species from step (i) to astandard control representing the amount of the RNA species in thecorresponding sample from an average pregnant woman with a chromosomallynormal fetus, wherein an increase or a decrease in the amount of RNAspecies from the standard control indicates an increased risk of havinga fetus with trisomy
 21. 28. The method of claim 27, wherein the RNAspecies is derived from ADAMTS1, APP, ATP5O, EFEMP1, or TFRC, and anincrease in the amount of the RNA species from the standard controlindicates an increased risk of having a fetus with trisomy
 21. 29. Themethod of claim 27, wherein step (i) comprises using a reversetranscriptase polymerase chain reaction (RT-PCR).
 30. The method ofclaim 29, wherein step (i) further comprises using mass spectrometry.31. The method of claim 27, wherein step (i) comprises using apolynucleotide hybridization method.
 32. The method of claim 27, whereinstep (i) comprises using a primer extension reaction.
 33. The method ofclaim 27, wherein the woman is during the first trimester of gestation.34. The method of claim 27, wherein the woman is during the second orthird trimester of gestation.
 35. The method of claim 27, wherein theblood is plasma.
 36. The method of claim 27, wherein the blood is serum.37. The method of claim 27, wherein the increase in the amount of theRNA species from the standard control is more than 2-fold.
 38. Themethod of claim 27, wherein the decrease in the amount of the RNAspecies from the standard control is more than 50%.
 39. A kit fordetecting the presence of a fetus with trisomy 21 in a pregnant woman,the kit comprising: (i) PCR primers for quantitatively determining theamount of one or more RNA species in a biological sample obtained fromthe pregnant woman, wherein the RNA species is independently selectedfrom RNA species derived from genetic loci consisting of COL6A1, COL6A2,SOD1, APP, BTG3, ATP5J, ADAMTS1, BACE2, DSCR5, ITSN1, PLAC4, ATP5O,LOC90625, EFEMP1, and TFRC, and wherein the biological sample is blood,washing from the reproductive tract, amniotic fluid, urine, saliva, orchorionic villus; and (ii) a standard control representing the amount ofthe RNA species in the corresponding sample from an average pregnantwoman with a chromosomally normal fetus.
 40. A method for detectingpregnancy in a woman, the method comprising: (i) quantitativelydetermining the amount of one or more RNA species in a biological sampleobtained from the woman, wherein the RNA species is independentlyselected from RNA species derived from genetic loci consisting ofCOL6A1, COL6A2, SOD1, ATP5O, ADAMTS1, DSCR5, and PLAC4, and wherein thebiological sample is blood, washing from the reproductive tract,amniotic fluid, urine, saliva, or chorionic villus; and (ii) comparingthe amount of the RNA species from step (i) to a standard controlrepresenting the amount of the RNA species in the corresponding samplefrom an average non-pregnant woman, wherein an increase or decrease inthe amount of the RNA species from the standard control indicatespregnancy.
 41. The method of claim 40, wherein the RNA species isderived from COL6A1, COL6A2, ATP5O, or PLAC4, and wherein an increase inthe amount of the RNA species from the standard control indicatespregnancy.
 42. The method of claim 40, wherein step (i) comprises usinga reverse transcriptase polymerase chain reaction (RT-PCR).
 43. Themethod of claim 42, wherein step (i) further comprises using massspectrometry.
 44. The method of claim 40, wherein step (i) comprisesusing a polynucleotide hybridization method.
 45. The method of claim 40,wherein step (i) comprises using a primer extension reaction.
 46. Themethod of claim 40, wherein the woman is during the first trimester ofgestation.
 47. The method of claim 40, wherein the woman is during thesecond or third trimester of gestation.
 48. The method of claim 40,wherein the blood is plasma.
 49. The method of claim 40, wherein theblood is serum.
 50. The method of claim 40, wherein the increase in theamount of RNA from the standard control is more than 2-fold.
 51. Themethod of claim 40, wherein the decrease in the amount of RNA from thestandard control is more than 50%.
 52. A kit for detecting pregnancy ina woman, the kit comprising: (i) PCR primers for quantitativelydetermining the amount of one or more RNA species in a biological sampleobtained from the woman, wherein the RNA species is independentlyselected from RNA species derived from genetic loci consisting ofCOL6A1, COL6A2, SOD1, ATP5O, ADAMTS1, DSCR5, and PLAC4, and wherein thebiological sample is blood, washing from the reproductive tract,amniotic fluid, urine, saliva, or chorionic villus; and (ii) a standardcontrol representing the amount of the RNA species in the correspondingsample from an average non-pregnant woman.